Use of the monoclonal antibody DAKO-ERbeta (8D5-1) to measure oestrogen receptor beta in breast cancer cells

BACKGROUND: Following a lethal injury, two modes of cell death can be distinguished, apoptosis and primary necrosis. Cells pass through a prelethal stage characterized by a preservation of membrane integrity, in which they shrink (apoptosis) or swell (oncosis, the early phase of primary necrosis). During apoptosis, a loss of phospholipid asymmetry leads to exposure of phosphatidylserine (PS) residues on the outer leaflet of the plasma membrane. We examined whether the external PS exposure, initially supposed to be specific for apoptosis, was also observed in oncotic cells. METHODS: Human peripheral lymphocytes, Jurkat T cells, U937 cells, or HeLa cells were submitted to either apoptotic or oncotic stimuli. PS external exposure was assessed after binding of FITC-conjugated annexin V as was the loss of membrane integrity after propidium iodide (PI) uptake. Morphological examination was performed by optical or electron microscopy. RESULTS: Similarly to apoptotic cells, oncotic cells expose external PS residues while preserving membrane integrity. Consequently, oncotic cells exhibit the annexin V+ PI- phenotype, previously considered to be specific for apoptotic cells. CONCLUSIONS: This study concludes that the annexin V/PI assay does not discriminate between apoptosis and oncosis and that it can be a useful tool to study oncosis by flow cytometry.     

体外培养脐血单个核细胞与CD34+富集细胞

对比MNC和CD34 +富集细胞在SCF +IL 3+IL 6 +FL +Tpo细胞因子组合下的体外扩增特性 ,发现 :CD34 +富集细胞具有很高的扩增潜力 ,在本实验条件下其总细胞持续扩增了 8周 ,扩增倍数达 312 70 9± 86 40 5倍 ;而MNC在培养至第 4周扩增就已呈现下降趋势 ,最大仅扩增了 5 3 3± 6 2倍。对比集落和CD34 +细胞的扩增发现 ,MNC的集落密度和CD34 +细胞含量由第 0天至第 7天有一个上升的过程 ,而CD34 +富集细胞在培养过程中 ,集落密度和CD34 +细胞含量却始终呈下降趋势。在体外培养过程中 ,CD34 +富集细胞的CFU GM和CD34 +细胞最大分别扩增了 185 7± 14 1和 191 7± 188 8倍 ,明显高于MNC的 12 4± 3 2和 5 0 6± 33 2倍 ;而CD34 +富集细胞和MNC的BFU E则只实现了少量扩增 ,分别为 7 2± 5 2和 10 1± 3 4倍。结果显示 ,从CD34 +富集细胞出发扩增造血干 祖细胞 ,可以得到更多的CD34 +细胞和CFU GM集落形成细胞     

A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5-triphosphate phosphatase activity

The Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) is a potent immunotoxin that induces G(2) arrest in human lymphocytes. We now show that the CdtB subunit exhibits phosphatidylinositol (PI)-3,4,5-triphosphate phosphatase activity. Breakdown product analysis indicates that CdtB hydrolyzes PI-3,4,5-P(3) to PI-3,4-P(2) and therefore functions in a manner similar to phosphatidylinositol 5-phosphatases. Conserved amino acids critical to catalysis in this family of enzymes were mutated in the cdtB gene. The mutant proteins exhibit reduced phosphatase activity along with decreased ability to induce G(2) arrest. Consistent with this activity, Cdt induces time-dependent reduction of PI-3,4,5-P(3) in Jurkat cells. Lymphoid cells with defects in SHIP1 and/or ptase and tensin homolog deleted on chromosome 10 (PTEN) (such as Jurkat, CEM, Molt) and, concomitantly, elevated PI-3,4,5-P(3) levels were more sensitive to the toxin than HUT78 cells which contain functional levels of both enzymes and low levels of PI-3,4,5-P(3). Finally, reduction of Jurkat cell PI-3,4,5-P(3) synthesis using the PI3K inhibitors, wortmannin and LY290004, protects cells from toxin-induced cell cycle arrest. Collectively, these studies show that the CdtB not only exhibits PI-3,4,5-P(3) phosphatase activity, but also that toxicity in lymphocytes is related to this activity.     

Annexin-V promotes anti-tumor immunity and inhibits neuroblastoma growth in vivo

The goal of the current study is to determine the effects of blocking phosphatidylserine (PS) on the growth of neuroblastoma in mice. PS, an anionic phospholipid restricted to the cytoplasmic surface of plasma membranes in most cells, is externalized to the surface of apoptotic cells. PS has been shown to induce immune tolerance to self-antigens. PS can also be found on the surface of live cells and in particular tumor cells. Annexin-V (AnV) is a protein that specifically binds and blocks PS. To determine the effects of blocking PS with AnV on tumor growth and immunogenicity, mice were inoculated with AGN2a, a poorly immunogenic murine neuroblastoma that expresses high level of PS on the cell surface. Survival and anti-tumor T cell response were determined. AGN2a were engineered to secrete AnV. Secreted protein effectively blocked tumor PS. 40?% of mice inoculated with AnV-expressing AGN2a cells survived free of tumor, whereas none of the mice inoculated with control cells survived (p?=?0.0062). The benefits of AnV were lost when mice were depleted of T cells. The findings suggest that AnV could protect mice from tumor challenge through an immune mediated mechanism. Mice were then immunized with irradiated AnV-secreting or control cells, and challenged with wild-type AGN2a cells. AnV-secreting cell vaccine protected 80?% of mice from AGN2a challenge, while control cell vaccine prevented tumor growth in only 30?% of animals (p?=?0.012). ELISPOT analysis demonstrated that AnV-secreting cell vaccine induced a greater frequency of interferon-gamma producing splenic T cells. T cells isolated from mice immunized with AnV-secreting but not control vaccine lysed AGN2a. In summary, AnV blocked PS, enhanced T cell mediated tumor immunity, and inhibited tumor growth.     

Lineage depletion of stromal vascular fractions isolated from human adipose tissue: a novel approach towards cell enrichment technology

The therapeutic rationale for tissue repair and regeneration using stem cells is at its infancy and needs advancement in understanding the role of individual component’s innate capability. As stem cells of adipose tissue reside in a more heterogeneous population of stromal vascular fractions, cell separation or sorting becomes an eminent step towards revealing their unique properties. This study elucidates the comparative efficacy of lineage depleted adipose derived stromal vascular fraction (SVF) and their innate ability using magnetic activated cell sorter (MACS). To this end, isolated SVF from human adipose tissue was lineage depleted according to the manufacturer’s instructions using specific antibody cocktail through MACS. The enriched lineage negative (lin−) and lineage positive (lin+) cell fractions were cultured, phenotypically characterized for the panel of cell surface markers using flowcytometry and subjected to osteoblastic and adipogenic differentiation. The expression profile obtained for lin− cells was CD34−/CD45−/HLADR−/CD49d−/CD140b−/CD31−/CD90+/CD105+/CD73+/CD54+/CD166+/CD117− when compared to Lin+ cells expressing CD34+/CD45+/HLADR−/CD49d−/CD140b+/CD31−/CD90+/CD105+/CD73+/CD54+/CD166+/CD117+ (CD—cluster of differentiation). These results, thus, advances our understanding on the inherent property of the individual cell population. Furthermore, both the fractions exhibited mesodermal lineage differentiation capacity. To conclude, this research pursuit rationalized the regenerative therapeutic applicability of both lin− and lin+ cultures of human adipose tissue for disorders of mesodermal, haematological and vascular origin.     

Heterogeneity of the regulation of phospholipase C by phorbol esters in T lymphocytes

In many cells, protein kinase C (PKC) activation inhibits cellular phospholipase C thereby preventing receptor-mediated phosphatidylinositol (PI) metabolism. In T lymphocytes, the T cell antigen receptor (Ti)/CD3 complex regulates PI hydrolysis and we have examined the consequences of PKC activation on Ti/CD3-mediated PI metabolism in human peripheral blood-derived T lymphocytes (T lymphoblasts) and the leukemic T cell line Jurkat. In Jurkat cells, PI metabolism after Ti/CD3 stimulation, is inhibited by PKC activation. PKC activation also inhibits calcium-induced PI metabolism in permeabilized Jurkat cells. In marked contrast, PI metabolism after Ti/CD3 stimulation in T lymphoblasts, is not inhibited by PKC activation. Moreover, in permeabilized T lymphoblasts PI metabolism can be induced by calcium in synergy with guanine 5'-O-(3-thiotrisphosphate) via a PKC-insensitive mechanism. The different effect of PKC stimulation on PI metabolism in Jurkat cells and T lymphoblasts reveals heterogeneity of PLC regulation in T lymphocytes. The data also indicate that the role of PKC as a regulator of Ti/CD3 signal transduction can differ depending on cell type.     

白藜芦醇以Caspase依赖和非依赖两种方式导致Jurkat细胞死亡

应用形态学观察、流式细胞仪检测、Western印迹和DNA凝胶电泳等方法研究白藜芦醇对Jurkat细胞的作用。发现白藜芦醇处理组中细胞有皱缩、出泡、染色质边集等现象,但染色质浓缩呈散在团块状且不致密。细胞质结构疏松,线粒体肿胀,脊消失。少见凋亡小体。在白藜芦醇处理组,Western印迹可检测到弱的17kDacaspase-3条带,DNA凝胶电泳可以检测到梯状DNA和弥散条带;流式细胞仪在白藜芦醇处理组检测到大量PI单阳性细胞和少量膜联蛋白V单阳性细胞。Z-VAD-FMK干预后可以发现细胞死亡率降低,同时该组梯状DNA消失,但是大分子量弥散DNA条带依然可以检测到。结果表明白藜芦醇可以通过caspase依赖和非依赖途径导致Jurkat细胞死亡。此分子机制的明确将为白藜芦醇应用于临床白血病的治疗打下理论基础。     

Increased DNA single-strand break joining activity in UV-irradiated CD34+ versus CD34- bone marrow cells

The kinetics of UV-irradiation-induced (254 nm) DNA single-strand breaks (SSBs) were studied in single human hematopoietic cells using alkaline comet assay. Three cell populations were investigated: (i) Bone marrow mononuclear cells (BMMNCs) isolated by density gradient centrifugation, (ii) CD34- cells, and (iii) CD34+ cells. The two latter populations were purified from BMMNCs by negative and positive selection, respectively, using anti-CD34 immunobeads. SSBs were induced faster by 10 and 50 J/m2 than by 2 J/m2 and those caused by 2 J/m2 were joined faster that those caused by 10 or 50 J/m2. During the first 1.5 h after irradiation with a dose of 10 J/m2, CD34+ cells joined SSBs faster than did BMMNCs. The superior joining capacity of CD34+ cells was further substantiated with a higher UV dose. The comet lengths, indicating the extent of DNA repair, among 8/8 study subjects were shorter in CD34+ than in CD34- cells when assessed 24 h after a dose of 50 J/m2. Overall, the comet lengths at 24 h after irradiation were: CD34+ cells; 39+/-12 *m, and CD34- cells; 65+/-18 *m (8 subjects, 50 cells measured from each donor, mean+/-S.D.; p=0.0087, Mann-Whitney U-test). These results strongly suggest that nucleotide excision repair, the major mechanism responsible for the repair of UV-irradiation-induced DNA lesions in mammalian cells, is increased in CD34+ cells compared with CD34- cells and with BMMNCs. These results may have implications in stem cell purging, clinical chemotherapy and carcinogenesis.     

The CD24 antigen discriminates between pre-B and B cells in human bone marrow.

When bone marrow (BM) lymphoid cells from 12 adult healthy donors were labeled by CD24 antibodies and analyzed by flow cytometry, two positive populations of cells were demonstrated in each sample (by a separated bimodal specific immunofluorescence). One population had intermediate CD24-Ag density (termed CD24+ cells) whereas the other had high CD24-Ag density (termed CD24(2+) cells). CD24+ cells represented 5.8 +/- 2.7% of the total lymphoid BM cells and CD24(2+) cells 5.6 +/- 2.5%. Using dual fluorescence analysis on eight samples, all CD24+ cells expressed the CD21 and CD37 mature B cell Ag and also surface IgM (sIgM), but this population lacked CD10 Ag. These cells also expressed CD19 Ag, and at a higher density than CD24(2+) cells. They were also positive for HLA-DR Ag. Conversely, CD24(2+) cells were shown to be early cells of the B cell lineage. While all the CD24(2+) cells were HLA-DR+ and CD19+, 64 +/- 16% of them expressed CD20 Ag (at a lower density than CD24+ cells), 65 +/- 21% CD10 Ag, and 22 +/- 8% were positive for cytoplasmic mu-chains (c mu). None of these cells expressed the CD21 and CD37 mature B cell Ag or sIgM. Additional experiments on four different healthy donors demonstrated that 30 +/- 9% of the CD24(2+) cells expressed the CD34 Ag and that the CD24+ cells did not express it. Thus, the CD24 Ag permits discrimination between two populations of the B cell lineage present in adult BM: 1) A CD24(2+) cell population including "pre" pre-B cells (HLA-DR+, CD19+, CD10+/-, CD20-, CD21-, CD34+, CD37-, c mu-), "intermediate" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu-), and "true" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu+). 2) A CD24+ cell population including B cells of the standard phenotype (HLA-DR+, CD19+, CD10-, CD20+, CD21+, CD34-, CD37+, c mu-, sIgM+).     

Phosphatidylserine-dependent adhesion of T cells to endothelial cells

Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.     

Pre-clinical development of cord blood-derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine

BACKGROUND: We have previously demonstrated that the copper chelator tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells (CD34+CD38-, CD34+CD38-Lin-) in human umbilical cord blood (CB)-derived CD34+ cell cultures. This study extends our previous findings that copper chelation can modulate the balance between self-renewal and differentiation of hematopoietic progenitor cells. METHODS: In the present study we established a clinically applicative protocol for large-scale ex vivo expansion of CB-derived progenitors. Briefly, CD133+ cells, purified from CB using Miltenyi Biotec's (Bergisch Gladbach, Germany) CliniMACS separation device and the anti-CD133 reagent, were cultured for 3 weeks in a clinical-grade closed culture bag system, using the chelator-based technology in combination with early-acting cytokines (SCF, thrombopoietin, IL-6 and FLT-3 ligand). This protocol was evaluated using frozen units derived from accredited cord blood banks. RESULTS: Following 3 weeks of expansion under large-scale culture conditions that were suitable for clinical manufacturing, the median output value of CD34+ cells increase by 89-fold, CD34+CD38- increase by 30-fold and CFU cells (CFUc) by 172-fold over the input value. Transplantation into sublethally irradiated non-obese diabetic (NOD/SCID) mice indicated that the engraftment potential of the ex vivo expanded CD133+ cells was significantly superior to that of unexpanded cells: 60+/-5.5% vs. 21+/-3.5% CD45+ cells, P=0.001, and 11+/-1.8% vs. 4+/-0.68% CD45+CD34+ cells, P=0.012, n=32, respectively. DISCUSSION: Based on these large-scale experiments, the chelator-based ex vivo expansion technology is currently being tested in a phase 1 clinical trial in patients undergoing CB transplantation for hematological malignancies.     

The supportive effects of IL-7 on eosinophil progenitors from human bone marrow cells can be blocked by anti-IL-5.

Human rIL-7 was studied for its effects on myeloid and erythroid progenitors from human bone marrow cells. IL-7 did not support the granulocytic/monocytic or erythroid lineage but exclusively stimulated eosinophil colony formation (CFU-Eo) (4 +/- 3 vs 48 +/- 17 CFU-Eo/10(5) nonadherent fraction-non-T cell (NAF-NT) cells). This supportive effect was not mediated by T cells or monocytes because similar results were obtained with or without T cell or adherent depleted cell fractions. In addition, it was shown that CD34+ sorted cells could be stimulated by IL-7 (0 vs 15 +/- 9 CFU-Eo/3 x 10(3) CD34+ cells) Furthermore studies with IL-3 or granulocyte-macrophage CSF (GM-CSF) demonstrated an additive effect on the IL-7 supported colony formation. Finally, experiments were performed with anti-IL-3, anti-GM-CSF, anti-IL-1, and anti-IL-5 to exclude the possibility that IL-7 indirectly stimulated the eosinophil progenitor cell. Anti-GM-CSF, anti-IL-1, or anti-IL-3 did not influence the supportive effects of IL-7. However, anti-IL-5 did abolish the effects of IL-7 on the eosinophil colony formation (69 +/- 15 vs 3 +/- 2 CFU-Eo/10(5) NAF-NT, n = 3). Similar results were obtained with CD34+ sorted cells. Moreover, IL-5 mRNA expression could be demonstrated in IL-7-stimulated NAF-NT cells. These data suggest that the supportive effects of IL-7 on eosinophil precursors are mediated by the endogenous release of IL-5.     

Maturation-dependent adhesion of human B cell precursors to the bone marrow microenvironment

Murine B cell precursors can be induced to proliferate in culture if allowed to bind to bone marrow derived adherent cells prepared under specific conditions. We studied the binding of human B cell precursor subpopulations to various in vitro microenvironments to determine which conditions may potentially be suitable models for human B precursor differentiation. Using the markers CD10, CD34, and CD20, B lineage populations of increasing maturation were quantitated: CD10+/CD34+, CD10+/CD20-, CD10+/CD20+, and CD10-/CD20+ cells in marrow, and CD10-/CD20+ mature B cells in peripheral blood. The adhesion of subpopulations of blood and marrow-derived light density cells to adherent cell layers or matrix was studied following a 2-h incubation in 24-well plates. The absolute number of bound B lineage cells was determined by cell counts and flow cytometry analysis. The adherence of B lineage cells to passaged human marrow fibroblasts (BM-FB) was highest in the most immature CD10+/CD34+ cells (34.3 +/- 4.2%), decreasing steadily with each stage of maturation to the peripheral blood B cells (11.2 +/- 2.4%). Increased adhesion of CD10+ B cell precursors relative to CD10-/CD20+ marrow B cells was confirmed by adhesion studies using sorted cells. The two most immature B lineage cells (CD10+CD34+ and CD10+/CD20-) showed more adherence to BM-FB than any other cell type tested, except for monocytes. Only B lineage precursor cells, erythroid precursors and CD10-/CD34+ cells showed significantly greater binding to BM-FB than to plastic. B lineage precursors bound equally well to primary and passaged human marrow fibroblasts, but bound significantly less well to passaged human foreskin fibroblasts, primary human marrow stroma, extracellular matrix of marrow fibroblasts, or fibronectin. These results suggest that specific binding to marrow fibroblasts is part of the differentiation program of early B lineage precursors. This binding activity gradually and predictably decreases during B lineage differentiation, in contrast to expression of other binding receptors, such as LFA-1 and CD44, which increase during B lineage maturation.     

Flow cytometric detection of circulating dendritic cells in healthy subjects

Dendritic cells (DCs) are the key antigen-presenting cells controlling the initiation of the T cell- dependent immune response. Currently, two peripheral blood DC subsets have been identified on the basis of their CD11c expression. The CD11c-negative (CD11c-) DCs (expressing high levels of CD123) are designated as lymphoid-derived DCs (DC2), whereas the CD11c+/CD123- cells, do identify the myeloid-derived DCs (DC1). A growing number of studies have been conducted in recent years on both the quantitative and functional alterations of DCs and their subsets in different pathological conditions. In the present study we assessed, using two different flow cytometric (FCM) techniques, the normal profile of blood DCs in 50 italian adult healthy subjects (M/F: 25/25, median age 42.5 years, range 20-65). The percentage and the absolute number of DCs and their subsets, were obtained starting from whole blood samples in two ways: 1) by calculating the number of DCs when gated as lineage-negative/ HLA-DR+ and identifing the two subsets as CD11c+ (DC1) and CD123+ (DC2) and 2) by using three specific markers: BDCA.1 (CD11c+ high/CD123+ low, myeloid DCs); BDCA.2 (CD11c-/ CD123+high, lymphoid DCs); BDCA.3 (CD11c+low /CD123-, myeloid DCs). Six parameters, 4-color FCM analysis were perfomed with a BD FACSCanto equipment. The mean values of the percentage and of the absolute number were: 0.5+/-0.2% and 30+/-11 cells/microL for DCs; 0.2+/-0.1% and 15+/-6 cells/microL for DC1; 0.2+/-0.1% and 15+/-7 cells/microL for DC2. The same values were: 0.2+/-0.1% and 16+/-7 cells/microL for BDCA.1; 0.2+/-0.1% and 12+/-7 cells/microL for BDCA.2; 0.02+/-0.01% and 2+/-1 cells/microL for BDCA.3, respectively. Our study confirmes that the two types of FCM analysis are able to identify the DC population. We also provides the first reference values on normal rates and counts of blood DCs in italian adult healthy subjects.     

Single-cell image analysis to assess ABC-transporter-mediated efflux in highly purified hematopoietic progenitors

BACKGROUND: Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux. METHODS: We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter-mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively). RESULTS: The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein-mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 +/- 0.35 and 1.14 +/- 0.11, respectively; P = 0.01). P-glycoprotein-mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 +/- 0.19 and 1.28 +/- 0.18, respectively). CONCLUSION: The described method is a valuable tool for assessing ABC-transporter-mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells.     

Amoebic PI3K and PKC Is Required for Jurkat T Cell Death Induced by Entamoeba histolytica

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.     

A protein kinase C-activating phorbol ester accelerates the T cell antigen receptor-stimulated phosphatidylinositol cycle in normal human CD4+ T cells.

Ligation of the TCR on Jurkat T lymphoblastoid cells causes an 1,4,5-inositol trisphosphate-dependent rise in intracellular cytoplasmic calcium that is inhibited by PMA, a potent activator of protein kinase C. Consequently, protein kinase C is widely believed to mediate feedback inhibition of TCR-activated phospholipase C. We have now extended these studies to normal unblasted human CD4+ T lymphocytes, examining the PMA sensitivity of both the TCR complex-mediated release of total inositol-phosphates and the resynthesis of the parent phosphoinositides. In contrast to Jurkat, in which PMA inhibited release of 1,4,5-inositol trisphosphate by 60% and total inositolphosphates by 40% (50% inhibitory concentration, 5.6 nM), normal cells displayed a marked increase in anti-CD3-induced phosphatidylinositol (PI) cycling in the presence of PMA. Both total inositolphosphate release and PI resynthesis were maximally elevated (88% and 342%, respectively) by a PMA concentration that also optimally supported a subsequent proliferative response; the ED50 was at least 11.7-fold lower than that for the inhibitory effect of PMA on breakdown of total Jurkat PI. A PKC nonactivating phorbol ester had no effect. If anti-CD3 was replaced by the mitogenic lectin PHA, PI resynthesis was similarly up-regulated by PMA in these highly purified cells. The PMA up-regulatory phenomenon was not a simple consequence of cell blastogenesis, inasmuch as there was no early effect on the non-signaling-associated phosphatidylethanolamine compartment after CD3 stimulation. Thus, PKC activation appears to accelerate TCR-linked PI metabolism in normal Th cells, in contrast to the feedback inhibitor paradigm observed in Jurkat and other tumor cell systems.