技术资料

OBJECTIVE: To determine the response of bone marrow progenitor cells from patients with myelodysplastic syndromes (MDS) to culture in physiologic oxygen tension. METHODS: Methylcellulose progenitor assays using both unfractionated bone marrow mononuclear cells (MNCs) and purified CD34(+) progenitors were performed in atmospheric oxygen (18.6% O(2)) or one of two levels of hypoxia (1% and 3% O(2)). Assays were performed using normal donor marrow,MDS patient marrow,acute myelogenous leukemia marrow or peripheral blood blasts,chronic phase chronic myelogenous leukemia (CML) marrow MNCs,and blast crisis CML peripheral blood. RESULTS: The majority of MDS samples showed decreased colony-forming units (CFU) in 18.6% O(2) compared to normal controls,as expected. However,in either 1% or 3% O(2),9 of 13 MDS samples demonstrated augmentation of CFUs beyond that observed in normal controls,with 6 of 13 demonstrating a greater than ninefold augmentation. This effect is cell autonomous,as it persisted after purification of CD34(+) progenitor cells. Additionally,the augmented response to physiologic oxygen tension is specific to MDS,as it was not observed in either acute or chronic myelogenous leukemia samples. CONCLUSION: These results suggest that the reported decrease in MDS CFUs reflects greater sensitivity of MDS progenitors or their progeny to the nonphysiologic oxygen tensions routinely used in vitro,rather than a true decrease in progenitor frequency. Importantly,these experiments for the first time describe an experimental system that can be used to study the growth of primary cells from patients with MDS. View Publication

Mice lacking the zinc finger transcriptional repressor protein GFI-1 are neutropenic. These mice generate abnormal immature myeloid cells exhibiting characteristics of both macrophages and granulocytes. Furthermore,Gfi-1(-/-) mice are highly susceptible to bacterial infection. Interestingly,Gfi-1(-/-) myeloid cells overexpress target genes of the PU.1 transcription factor such as the macrophage colony-stimulating factor receptor and PU.1 itself. We therefore determined whether GFI-1 modulates the transcriptional activity of PU.1. Our data demonstrate that GFI-1 physically interacts with PU.1,repressing PU.1-dependent transcription. This repression is functionally significant,as GFI-1 blocked PU.1-induced macrophage differentiation of a multipotential hematopoietic progenitor cell line. Retroviral expression of GFI-1 in primary murine hematopoietic progenitors increased granulocyte differentiation at the expense of macrophage differentiation. We interbred Gfi-1(+/-) and PU.1(+/-) mice and observed that heterozygosity at the PU.1 locus partially rescued the Gfi-1(-/-) mixed myeloid lineage phenotype,but failed to restore granulocyte differentiation. Our data demonstrate that GFI-1 represses PU.1 activity and that lack of this repression in Gfi-1(-/-) myeloid cells contributes to the observed mixed lineage phenotype. View Publication

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified,biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes,a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts,including G6b,G6f,LRRC32,LAT2,and the G protein-coupled receptor SUCNR1,encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins,and flow cytometric analysis confirmed the expression of G6b,G6f,and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b,G6f,and LRRC32 are restricted to the platelet lineage,whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest,because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists. View Publication

An urgent need exists to devise strategies to augment antiviral immune responses in patients with HIV who are virologically well controlled and immunologically stable on highly active antiretroviral therapy (HAART). The objective of this study was to compare the immunomodulatory effects of the cytokines interleukin (IL)-21 with IL-15 on CD8 T cells in patients with HIV RNA of less than 50 copies/mL and CD4 counts greater than 200 cells/mm.(3) Patient CD8 T cells displayed skewed maturation and decreased perforin expression compared with healthy controls. Culture of freshly isolated patient peripheral-blood mononuclear cells (PBMCs) for 5 hours to 5 days with IL-21 resulted in up-regulation of perforin in CD8 T cells,including memory and effector subsets and virus-specific T cells. IL-21 did not induce T-cell activation or proliferation,nor did it augment T-cell receptor (TCR)-induced degranulation. Treatment of patient PBMCs with IL-15 resulted in induction of perforin in association with lymphocyte proliferation and augmentation of TCR-induced degranulation. Patient CD8 T cells were more responsive to cytokine effects than the cells of healthy volunteers. We conclude that CD8 T cells of patients with HIV can be modulated by IL-21 to increase perforin expression without undergoing overt cellular activation. IL-21 could potentially be useful for its perforin-enhancing properties in anti-HIV immunotherapy. View Publication

Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers,including myeloid malignancies. We investigated the effects of an oncogenic Kras(G12D) allele on phosphorylated signaling molecules in primary c-kit(+) lin(-/low) hematopoietic stem/progenitor cells. Comparison of wild-type and Kras(G12D) c-kit(+) lin(-/low) cells shows that K-Ras(G12D) expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5,ERK,and S6 under basal and stimulated conditions. Whereas Kras(G12D) cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor,we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell-targeted interventions. View Publication

OBJECTIVE Pancreatic cancer continues to be an urgent clinical problem. We used the novel DNA methyltransferase inhibitor Zebularine and the histone deacetylase inhibitor SAHA to investigate the epigenetic influence on viability and differentiation of the pancreatic cancer cell lines YAP C,DAN G and Panc-89 in vitro and in vivo. MATERIAL AND METHODS Cell vitality,proliferation and expression of PDX-1,cytokeratin 7 and 20,chromogranin A,vimentin,bax and bcl-2 were determined on the protein and mRNA level in vitro and in a subcutaneous xenograft model. RESULTS A time- and dose-dependent increase of apoptosis,paralleled by decreased proliferation,was observed after incubation with single agents or a combination therapy with lower concentrations. This was associated with up-regulation of pro-apoptotic bax and a phenotypic stabilization by the enhanced expression of cytokeratin 7. In vivo,growth of xenografts was delayed with the most pronounced effect in Panc-89 after 1 week of daily intraperitoneal injections of Zebularine paralleled with CK7 up-regulation and down-regulation of dedifferentiation markers. CONCLUSIONS Epigenetic modulation via inhibition of DNA methyltransferase and histone deacetylase induces apoptosis in human pancreatic cancer cells in vitro and delays xenograft growth in vivo,which is associated with a morphological/molecular phenotypic stabilization. These compounds may therefore be suitable as adjunctive therapeutic agents in the treatment of pancreatic cancer. View Publication

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin insensitive and was recently shown to regulate the prosurvival kinase AKT by phosphorylation on Ser473. We investigated the molecular effects of mTOR inhibition by the rapamycin derivatives (RDs) temsirolimus (CCI-779) and everolimus (RAD001) in acute myeloid leukemia (AML) cells. Unexpectedly,RDs not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased p70S6K,4EPB1 phosphorylation,and GLUT1 mRNA,but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-cyclins in AML cells. Similar observations were made in samples from patients with hematologic malignancies who received RDs in clinical studies. Our study provides the first evidence that rapamycin derivatives inhibit AKT signaling in primary AML cells both in vitro and in vivo,and supports the therapeutic potential of mTOR inhibition strategies in leukemias. View Publication

The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs),hESCs normally undergo high rates of spontaneous apoptosis and differentiation,making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However,despite the accumulation of p53,it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types,including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation. View Publication

Effects of lysophosphatidic acid (LPA),an extracellular phospholipid signal,on the proliferation of rat embryonic neural stem cells (NSCs) and their differentiation into microtubule-associated protein 2 (MAP2)-positive and choline acetyltransferase (ChAT)-positive,i.e. cholinergic-committed neurons,were observed in vitro by [(3)H]-thymidine incorporation,immunocytochemistry,Western blot and other techniques. The results showed that: (1) Lower concentrations of LPA (0.01˜1.0 mumol/L) dose-dependently enhanced the uptake of [(3)H]-thymidine by NSCs cultured in specific serum-free medium,indicating a significant promotive action of LPA on the proliferation of NSCs. (2) After fetal bovine serum which induces and commences the differentiation of NSCs,was used in the medium,the lower concentrations of LPA increased the percentages of both MAP2- and ChAT-immunoreactive neurons,with a peak at 0.1 mumol/L LPA in two cases. (3) The promotive effects of LPA on the differentiation of MAP2- and ChAT-positive neurons were also supported by the up-regulation of the expressions of both MAP2 and ChAT proteins detected by Western blot. (4) At the early phase of differentiation of NSCs,the cell migration and neurite extension were enhanced significantly by lower dosages of LPA under phase-contrast microscope. These results suggest that LPA within certain lower range of concentrations promotes the proliferation of NSCs and their differentiation into unspecific MAP2-positive and specific cholinergic-committed neurons,and also strengthens the migration and neurite extension of the newly-generated neuronal (and also glial as reported elsewhere) progenitors. View Publication

Interferon regulatory factor-1 (IRF-1) is an important transcription factor that mediates interferon-gamma (IFN-gamma)-induced cell-signaling events. In this study,we examined whether 17beta-estradiol alters IRF-1 in splenic lymphocytes,in view of the immunomodulatory effects of this natural female sex hormone including its ability to alter IFN-gamma levels. We find that IRF-1 expression is markedly downregulated in splenocytes or purified T-cells from estrogen-treated mice at all time points studied when compared with their placebo counterparts. This decrease in IRF-1 in splenocytes from estrogen-treated mice is neither due to upregulation of IRF-1-interfering proteins (nucleophosmin or signal transducer and activator of transcription (STAT)-5) nor due to alternatively spliced IRF-1 mRNA. Given that IFN-gamma is a potent inducer of IRF-1,direct addition of recombinant IFN-gamma to splenocytes from either wild-type or IFN-gamma-knockout mice,or the addition of recombinant IFN-gamma to purified T-cells,was expected to stimulate IRF-1 expression. However,robust expression of IRF-1 in cells from estrogen-treated mice was not seen,unlike what was observed in cells from placebo-treated mice. Diminished IFN-gamma induction of IRF-1 in cells from estrogen-treated mice was noticed despite comparable phosphorylated STAT-1 activation. These studies are the first to show that estrogen regulates IFN-gamma-inducible IRF-1 in lymphoid cells,a finding that may have implications to IFN-gamma-regulated immune and vascular diseases. View Publication

In vivo endothelial commitment of adipose stem cells (ASCs) has scarcely been reported,and controversy remains on the contribution of ASCs to vascularization. We address the epigenetic commitment of ASCs to the endothelial lineage. We report a bisulfite sequencing analysis of CpG methylation in the promoters of two endothelial-cell-specific genes,CD31 and CD144,in freshly isolated and in cultures of ASCs before and after induction of endothelial differentiation. In contrast to adipose tissue-derived endothelial (CD31(+)) cells,freshly isolated ASCs display a heavily methylated CD31 promoter and a mosaically methylated CD144 promoter despite basal transcription of both genes. Methylation state of both promoters remains globally stable upon culture. Endothelial stimulation of ASCs in methylcellulose elicits phenotypic changes,marginal upregulation of CD31,and CD144 expression and restrictive induction of a CD31(+)CD144(+) immunophenotype. These events are accompanied by discrete changes in CpG methylation in CD31 and CD144 promoters; however,no global demethylation that marks CD31(+) cells and human umbilical vein endothelial cells occurs. Immunoselection of CD31(+) cells after endothelial stimulation reveals consistent demethylation of one CpG immediately 3' of the transcription start site of the CD31 promoter. Adipogenic or osteogenic differentiation maintains CD31 and CD144 methylation patterns of undifferentiated cells. Methylation profiles of CD31 and CD144 promoters suggest a limited commitment of ASCs to the endothelial lineage. This contrasts with the reported hypomethylation of adipogenic promoters,which reflects a propensity of ASCs toward adipogenic differentiation. Analysis of CpG methylation at lineage-specific promoters provides a robust assessment of epigenetic commitment of stem cells to a specific lineage. View Publication

Human embryonic stem cells (hESCs) provide great opportunities for regenerative medicine,pharmacological and toxicological investigation,and the study of human embryonic development. These applications require proper derivation,maintenance,and extensive characterization of undifferentiated cells before being used for differentiation into cells of interest. Undifferentiated hESCs possess several unique features,including their extensive proliferation capacity in the undifferentiated state,ability to maintain a normal karyotype after long-term culture,expression of markers characteristic of stem cells,high constitutive telomerase activity,and capacity to differentiate into essentially all somatic cell types. This chapter will summarize the current development in culture conditions and provide technical details for the evaluation and characterization of hESCs. View Publication