A model for the differentiation of human natural killer cells. Studies on the in vitro activation of Leu-11+ granular lymphocytes with a natural killer-sensitive tumor cell, K562.

A subpopulation of low density granular lymphocytes that express the natural killer (NK) cell-associated Leu-11 antigen (IgG Fc receptor) were stimulated directly by coculture with an NK-sensitive tumor cell, K562. T lymphocytes (Leu-11-) responded only weakly when cocultured with K562. The response of Leu-11+ cells apparently did not require exogeneous factors or accessory cells. The K562-activated cells retained expression of Leu-11 antigen, acquired activation antigens, and were highly cytotoxic against NK-sensitive and -insensitive tumor cells. Anti-IL-2 receptor monoclonal antibody minimally inhibited the activation of Leu-11+ cells by K562, but completely inhibited the phytohemagglutinin-induced activation of the Leu-11- cells from the same individual. Leu-11+ cells can be divided into Leu-7-11+ and Leu-7+11+ subpopulations using anti-Leu-7 antibody. These subsets were separated by two-color fluorescence-activated cell sorting and cocultured with K562. Proliferation by Leu-7-11+ cells was significantly greater than by Leu-11+7+ cells. Leu-7+11- granular lymphocytes and T lymphocytes (Leu-7-11-) typically proliferated only weakly when cocultured with K562. A proportion of the Leu-7-11+ cells acquired Leu-7 antigen after stimulation with K562, whereas the phenotype of Leu-7+11+, Leu-7+11-, and Leu-7-11- subsets was unaffected. These results demonstrate a developmental relationship between the Leu-7-11+ and Leu-7+11+ lymphocytes and suggest that Leu-7 antigen may be expressed late in the differentiation pathway of NK cells. The direct activation of highly purified Leu-11+ cells by coculture with K562 provides an in vitro model with which to study the activation and maturation of human NK cells

Interleukin 2 and stimulator lymphoblastoid cells will induce human thymocytes to bind and kill K562 targets.

Human thymocytes cultured in the presence of IL-2 and an irradiated B cell line became cytotoxic to K562 target cells. Thymocytes cultured alone or with only IL-2 exhibited almost no killing, but thymocytes cultured in the presence of stimulator cells alone exhibited low levels of cytotoxic activity. Removal of Fc gamma receptor-bearing cells from the activated thymocyte population almost completely abolished the binding and lytic activity. Separation of thymocytes into Fc microns+ and Fc microns-cells before culturing with IL-2 and stimulator cells revealed that only the Fc microns+ subpopulation developed into K562 killer cells. These findings indicate that modulation of Fc microns to Fc gamma receptors on the thymocyte cell surface is part of the maturation process of this particular subset of cytotoxic cells. Morphologically, most of the activated Fc gamma+ K562-binding cells were large, granulated lymphocytes. Only very few of the round, nongranulated small thymocytes were bound to K562 target cells

A flow cytometric study of the membrane potential of natural killer and k562 cells during the cytotoxic process

This study demonstrates that it is possible to investigate the membrane potential of interacting cells during the cytotoxic process using flow cytometry. Changes in the membrane potential of NK and K562 cells, involved in a cell-mediated cytotoxic process, were studied by standard and slit-scan flow cytometry, using the membrane potential sensitive fluorescent probe DiBAC4(3). The NK cells were labeled with a membrane marker (TR-18 or DiI) prior to incubation with K562 cells and the conjugates that were formed could be identified on the basis of the membrane marker fluorescence and light scattering signals. With a slit-scan technique we measured the membrane potential of each cell in a conjugate separately. The results show that depolarization of the K562 cell occurs as a consequence of the cytotoxic activity of the NK cell. This depolarization appears to be an early sign of cell damage because the cell membrane still remains impermeable to propidium iodide. Our data also indicate that depolarization of the NK cell occurs as a result of its cytotoxic activity

Single-cell epigenomic variability reveals functional cancer heterogeneity.

BackgroundCell-to-cell heterogeneity is a major driver of cancer evolution, progression, and emergence of drug resistance. Epigenomic variation at the single-cell level can rapidly create cancer heterogeneity but is difficult to detect and assess functionally.ResultsWe develop a strategy to bridge the gap between measurement and function in single-cell epigenomics. Using single-cell chromatin accessibility and RNA-seq data in K562 leukemic cells, we identify the cell surface marker CD24 as co-varying with chromatin accessibility changes linked to GATA transcription factors in single cells. Fluorescence-activated cell sorting of CD24 high versus low cells prospectively isolated GATA1 and GATA2 high versus low cells. GATA high versus low cells express differential gene regulatory networks, differential sensitivity to the drug imatinib mesylate, and differential self-renewal capacity. Lineage tracing experiments show that GATA/CD24hi cells have the capability to rapidly reconstitute the heterogeneity within the entire starting population, suggesting that GATA expression levels drive a phenotypically relevant source of epigenomic plasticity.ConclusionSingle-cell chromatin accessibility can guide prospective characterization of cancer heterogeneity. Epigenomic subpopulations in cancer impact drug sensitivity and the clonal dynamics of cancer evolution

CTCF-mediated transcriptional regulation through cell type-specific chromosome organization in the {\beta}-globin locus

The principles underlying the architectural landscape of chromatin beyond the nucleosome level in living cells remains largely unknown despite its potential to play a role in mammalian gene regulation. We investigated the 3-dimensional folding of a 1 Mbp region of human chromosome 11 containing the {\beta}-globin genes by integrating looping interactions of the insulator protein CTCF determined comprehensively by chromosome conformation capture (3C) into a polymer model of chromatin. We find that CTCF-mediated cell type specific interactions in erythroid cells are organized to favor contacts known to occur in vivo between the {\beta}-globin locus control region (LCR) and genes. In these cells, the modeled {\beta}-globin domain folds into a globule with the LCR and the active globin genes on the periphery. By contrast, in non-erythroid cells, the globule is less compact with few but dominant CTCF interactions driving the genes away from the LCR. This leads to a decrease in contact frequencies that can exceed 1000-fold depending on the stiffness of the chromatin and the exact positioning of the genes. Our findings show that an ensemble of CTCF contacts functionally affects spatial distances between control elements and target genes contributing to chromosomal organization required for transcription.Comment: Full article, including Supp. Mat., is available at Nucleic Acids Research, doi: 10.1093/nar/gks53

Down-regulatory mechanism of mammea E/BB from Mammea siamensis seed extract on Wilms' Tumor 1 expression in K562 cells.

BackgroundWilms' tumor 1 (WT1) is a biological marker for predicting leukemia progression. In this study, mammea E/BB, an active compound from Saraphi (Mammea siamensis) seed extract was examined for its effect on down-regulatory mechanism of WT1 gene expression, WT1 protein and mRNA stability, and cell proliferation in K562 cell line.MethodsM. siamensis seeds were obtained from the region of Chiang Mai (North of Thailand). Mammea E/BB was extracted from seeds of M. siamensis. WT1 protein expression and stability were evaluated by Western blot analysis. WT1 mRNA stability was assessed by qRT-PCR. WT1-DNA binding and WT1 promoter activity were assayed by ChIP assay and luciferase-reporter assay, respectively. Cell cycle arrest was studied by flow cytometry.ResultsTreatment with mammea E/BB led to down-regulation of WT1 expression. The suppression of WT1 expression did not involve protein and mRNA degradation. Rather, WT1 protein was down-regulated through disruption of transcriptional auto-regulation of the WT1 gene. Mammea E/BB inhibited WT1-DNA binding at the WT1 promoter and decreased luciferase activity. It also disrupted c-Fos/AP-1 binding to the WT1 promoter via ERK1/2 signaling pathway and induced S phase cell cycle arrest in K562 cells.ConclusionMammea E/BB had pleotropic effects on kinase signaling pathways, resulting in inhibition of leukemia cell proliferation

Density alteration in non-physiological cells

In the present study an important phenomenon of cells was discovered: the change of intracellular density in cell's response to drug and environmental factors. For convenience, this phenomenon is named as "density alteration in non-physiological cells" ( DANCE). DANCE was determined by discontinuous sucrose gradient centrifugation (DSGC), in which cells were separated into several bands. The number and position of the bands in DSGC varied with the change of cell culture conditions, drugs, and physical process, indicating that cell's response to these factors was associated with alteration of intracellular density. Our results showed that the bands of cells were molecularly different from each other, such as the expression of some mRNAs. For most cells tested, intracellular density usually decreased when the cells were in bad conditions, in presence of drugs, or undergoing pathological changes. However, unlike other tissue cells, brain cells showed increased intracellular density in 24 hrs after the animal death. In addition, DANCE was found to be related to drug resistance, with higher drug-resistance in cells of lower intracellular density. Further study found that DANCE also occurred in microorganisms including bacteria and fungus, suggesting that DANCE might be a sensitive and general response of cells to drugs and environmental change. The mechanisms for DANCE are not clear. Based on our study the following causes were hypothesized: change of metabolism mode, change of cell membrane function, and pathological change. DANCE could be important in medical and biological sciences. Study of DANCE might be helpful to the understanding of drug resistance, development of new drugs, separation of new subtypes from a cell population, forensic analysis, and importantly, discovery of new physiological or pathological properties of cells

Apoptotic effects of resveratrol, a grape polyphenol, on imatinib-sensitive and resistant K562 chronic myeloid leukemia cells

To examine the antiproliferative and apoptotic effects of resveratrol on imatinib-sensitive and imatinib-resistant K562 chronic myeloid leukemia cells. Antiproliferative effects of resveratrol were determined by the 3-Bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) cell proliferation assay. Apoptotic effects of resveratrol on sensitive K562 and resistant K562/IMA-3 cells were determined through changes in caspase-3 activity, loss of mitochondrial membrane potential (MMP), and apoptosis by annexin V-(FITC). The concentrations of resveratrol that inhibited cell growth by 50% (IC(50)) were calculated as 85 and 122 μM for K562 and K562/IMA-3 cells, respectively. There were 1.91-, 7.42- and 14.73-fold increases in loss of MMP in K562 cells treated with 10, 50, and 100 μM resveratrol, respectively. The same concentrations of resveratrol resulted in 2.21-, 3.30- and 7.65-fold increases in loss of MMP in K562/IMA-3 cells. Caspase-3 activity increased 1.04-, 2.77- and 4.8-fold in K562 and 1.02-, 1.41- and 3.46-fold in K562/IMA-3 cells in response to the same concentrations of resveratrol, respectively. Apoptosis was induced in 58.7%- and 43.3% of K562 and K562/IMA-3 cells, respectively, in response to 100 μM resveratrol. Taken together these results may suggest potential use of resveratrol in CML, as well as in patients with primary and/or acquired resistance to imatinib