CFTR in K562 human leukemic cells

In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 microM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.     

Glycophorins of human erythroleukemic K562 cells

Glycophorins related to alpha glycophorin, of the human erythrocyte membrane, were isolated from human erythroleukemic K562 cells. The glycophorins were purified using sodium dodecyl sulfate (SDS)/trichloroacetic acid fractionation and Folch and hot phenol extractions. 0.1-0.2 micrograms was obtained/10(8) cells, or approximately a 15% yield. SDS-gel electrophoresis revealed a pattern similar to erythrocyte alpha glycophorin except for the slower mobility of the glycophorin monomer. Two populations of K562 glycophorins, present in nearly equivalent amounts, were distinguished by their binding to Lens culinaris lectin agarose. The two populations exhibited similar gel electrophoretic patterns except for the presence of delta-like glycophorin exclusively in the population that did not bind to L. culinaris lectin. Immunoblotting revealed a lack of reaction of the major alpha and delta-like glycophorin bands in all K562 glycophorins with M or N erythrocyte glycophorin-specific monoclonal antibodies. Only minor species of intermediate electrophoretic mobility in glycophorins not binding to L. culinaris showed a reaction with these antibodies. Both populations of glycophorins incorporated radiolabeled glucosamine, mannose, and fucose and contained O-glycosidically linked tri- and tetrasaccharides, present in a ratio of approximately 1:1 indicating a significant degree of hyposialylation when compared to erythrocyte alpha glycophorin. No precursor/product relationship was demonstrated between the major forms of two populations. K562 cell surface labeling with lactoperoxidase revealed that only the glycophorins that exhibited binding to L. culinaris were accessible to iodination and could be the only species expressed at the cell surface.     

Renewal rates of murine T-lymphocyte subsets

The proliferative dynamics of T-cell subsets in lymphoid organs of BALB/c mice have been evaluated by means of [3H]thymidine injections for up to 3 days, followed by FACS and autoradiography. In the thymus, cells positive for both CD4 and CD8 antigens were more rapidly renewed than single-positive cells. Cell populations showing strong expression of Thy-1 and weak expression of CD5 antigens were more rapidly renewed than populations with the opposite antigen expressions. The results obtained from the spleen and the lymph nodes indicated that the majority of T cells have life spans exceeding a few days, and comparable cell kinetics were found in the different T-cell subpopulations. Results obtained after injections of hydroxyurea, which causes elimination of cycling cells, paralleled the autoradiographic results.     

Adriamycin resistance is characterized by ultrastructural changes in human leukaemic K562 cells in vitro.

Ultrastructural changes associated with adriamycin (ADM) resistance have been investigated in the human K562 leukaemic cell line: sensitive K562 cells, a resistant subline cultured in the continuous presence of ADM and resistant cells without ADM Transmission electron microscopy (TEM) study revealed that K562-resistant cells displayed ultrastructural modifications of the cell surface, chromatin and nucleolus conformation. Alterations were not directly related to the presence of adriamycin as deprivated cells exhibited modificated characters through a slow progressive recovery phenomenon.     

In vitro generation of human cells with cancer stem cell properties

Cancer stem cells (CSCs) have been implicated in the maintenance and progression of several types of cancer. The origin and cellular properties of human CSCs are poorly characterized. Here we show that CSC-like cells can be generated in vitro by oncogenic reprogramming of human somatic cells during neoplastic transformation. We find that in vitro transformation confers stem-cell properties to primary differentiated fibroblasts, including the ability to self-renew and to differentiate along multiple lineages. Tumours induced by transformed fibroblasts are hierarchically organized, and the cells that act as CSCs to initiate and maintain tumour growth are marked by the stage-specific embryonic antigen SSEA-1. Heterogeneous lineages of cancer cells in the bulk of the tumour arise through differentiation of SSEA-1(+) fibroblasts, and differentiation is associated with loss of tumorigenic potential. These findings establish an experimental system to characterize cellular and molecular properties of human CSCs and demonstrate that somatic cells have the potential to de-differentiate and acquire properties of CSCs.     

Biogenesis of glycophorin A in K562 human erythroleukemia cells

A monoclonal antibody (mAb-233) directed against an epitope in the nonglycosylated carboxyl-terminal region of human erythrocyte glycophorin A (GPA) was used in combination with metabolic labeling, the modification of N- and O-linked oligosaccharide processing by tunicamycin and monensin, and digestions with neuraminidase and O-glycanase to elucidate the pathway of GPA biogenesis in K562 human erythroleukemia cells. Cell-surface GPA is derived from two obligatory precursors in a stepwise manner. The initial GPA precursor has a Mr of 27,000 and appears to contain one N-linked high mannose oligosaccharide chain. In tunicamycin-treated cells, the initial precursor is similar in size (Mr = 24,000) to deglycosylated GPA from human erythrocytes. The 27-kDa initial precursor is rapidly converted to a transient 31-kDa intermediate by the addition of N-acetylgalactosamine residues to serine/threonine hydroxyl groups. Subsequent maturation involves the conversion of the high mannose chain to a complex-type oligosaccharide and the concomitant addition of galactose and sialic acid to internal N-acetylgalactosamine residues to extend the O-linked chains. These results define a single, stepwise processing pathway for the generation of all cell-surface GPA molecules and document for the first time the occurrence of both a unique initial precursor that contains a high mannose N-linked oligosaccharide chain but no O-linked sugars and a transient intermediate that appears to contain the same N-linked group and N-acetylgalactosamine at multiple serine/threonine residues. The properties of the intracellular GPA precursors and the relatively simple nature of the processing pathway reported herein contrast markedly with the characteristics of three intermediates and the complexity of two independent pathways in previously postulated schemes for GPA biogenesis (Gahmberg, C. G., Jokinen, M., Karhi, K. K., Kampe, O., Peterson, P. A., and Andersson, L. C. (1983) Methods Enzymol. 96, 281-298; Jokinen, M., Andersson, L. C., and Gahmberg, C. G. (1985) J. Biol. Chem. 260, 11314-11321).     

In vitro dendritic cell generation and lymphocyte subsets in myeloma patients: influence of thalidomide and high-dose chemotherapy treatment

While vaccination with antigen-pulsed dendritic cells (DCs) represents a promising therapeutic strategy in multiple myeloma (MM), clinical benefit, so far, has been limited to individual patients. To identify potential problems with this approach, we have analyzed the influence of treatment parameters, in particular high-dose chemotherapy (HD-CTX) and thalidomide, on in vitro DC generation and peripheral blood lymphocyte subsets in MM patients. From a total of 25 MM patients, including 14 patients on thalidomide treatment and 11 after HD-CTX, in vitro DC generation from peripheral blood monocytes under serum-free condition was investigated. In addition, peripheral blood lymphocyte subsets were assessed in 17 patients including 10 patients on thalidomide treatment and 9 patients after HD-CTX. Efficient in vitro generation of DCs (median 7.1×10⁶/100 ml peripheral blood; range 0.1–42.5×10⁶/100 ml peripheral blood) expressing DC-typical surface markers was observed in 23 MM patients (92%), although reduced expression of CD1a, CD40, CD83, and HLA-DR was observed in patients treated with thalidomide. With respect to lymphocyte subsets, MM patients showed significantly (p<0.05) reduced B and CD4+ lymphocytes in the peripheral blood. This effect was most prominent within 6 months of HD-CTX and in patients receiving thalidomide (usually in combination with CTX). CD8+ lymphocytes were significantly increased in MM patients. Thus, despite the well-known deficiencies in their immune system, adequate numbers of DCs can be generated in most myeloma patients. In patients treated with thalidomide, however, it remains to be seen whether the reduced expression of co-stimulatory molecules has functional relevance.The study was supported by Internes Forschungsförderungsprogramm Essen (IFORES) and Förderverein Essener Tumorklinik e. V.     

In vitro generation of high-titer prions

Prions are composed mainly, if not entirely, of PrP(Sc), an infectious misfolded isoform of PrP(C), the normal isoform of the prion protein. Here we show that protein misfolding cyclic amplification (PMCA)-generated hypertransmissible mink encephalopathy (HY TME) PrP(Sc) is highly infectious and has a titer that is similar, if not identical, to that associated with brain tissue from animals infected with the HY TME agent that are in the terminal stage of disease. These data demonstrate that PMCA efficiently replicates the prion agent and provide further support for the hypothesis that in vitro-generated prions are bona fide and are not due to contamination.     

Store-operated cationic channels in human myeloid leukaemia K562 cells

Using the whole-cell patch clamp technique, single channels operated by intracellular Ca(2+)-store depletion were first revealed in human myeloid leukaemia cells K562. A single store-operated channel could be detected in divalent-free extracellular solutions with Na+ as a permeant ion, and intracellular solutions with strong Ca(2+)-helating agent with some delay after whole-cell formation. Addition of inositol-1,4,5-triphosphate to the pipette solution resulted in a significant decrease of this latency. These channels had a conductance of 29 pS, and were inhibited by low concentration of external Ca2+. Our results enable us to assume that the revealed channels are calcium release-activated calcium channels, operated by Ca2+ depletion of endoplasmic reticulum.     

In vitro expansion of Ag-specific T cells by HLA-A*0201-transfected K562 cells for immune monitoring

BACKGROUND: Development of a practical and sensitive assay for evaluating immune responses against cancer Ag has been a challenge for immune monitoring of patients. We have established a reproducible method using peptide-pulsed K562-A*0201 cells as APC to expand Ag-specific T cells in vitro. This method may be applied for monitoring T-cell responses in cancer immunotherapy clinical trials. METHODS: Autologous PBMC from HLA-A*0201+ healthy donors and patients with melanoma were stimulated with peptide-pulsed K562-A*0201 cells under varying conditions. We investigated (1) different culture conditions, including the requirements for serum and cytokines for expansion of CD8+ T lymphocytes; (2) a range of peptide concentrations for Ag loading; (3) phenotypic characterization of responding T cells; and (4) APC:responder ratios and their effects on T-cell expansion. We validated these conditions by ELISPOT and intracellular cytokine staining (ICS) assays using peptides from influenza, Epslein-Barr Virus (EBV) and tyrosinase. RESULTS: Conditions for optimal T-cell expansion using K562-A*0201 APC included input of 2 x 10(6) PBMC, a 10 microg/mL peptide concentration to pulse K562-A*0201 cells, a 1:30 APC:responder T-cell ratio and culture in 10% autologous plasma supplemented with IL-2 and IL-15. In these conditions, Ag-specific T cells expanded >100-fold over a 10-day culture period (peak at day 12). DISCUSSION: This bulk culture method is simple and reliable for expanding human Ag-specific T cells using peptide-pulsed K562-A*0201 cells. This HLA-matched APC line can be adapted to other HLA haplotypes, and has advantages for monitoring clinical trials of immunotherapy with limited availability of autologous APC and PBMC from patients.     

Cartilage polysaccharide induces apoptosis in human leukemia K562 cells

In this study, we extracted a polysaccharide (short-chain polysaccharide [PS]) from porcine cartilage and examined its function in chronic myeloid leukaemia by using human K562 cells and mouse L1210 cells. Results of cell proliferation assay indicated that PS inhibited cancer cell growth at different concentrations, while it had little effect on normal cells. The presence of morphological aspects of apoptosis, such as nuclear shrinkage, was shown in H&E stained sections. The occurrence of PS-induced apoptosis was confirmed by TUNEL assay and cell cycle analysis. The results of immunofluorescent staining indicated the molecular mechanism underlying. Through interfering with the cell cycle of tumor cells, PS may induce apoptosis by downregulating the expression level of cyclin D1 and upregulating the level of p21 protein. Correlation analysis of apoptosis and MAPK suggested that inactivation of ERK was crucial for PS induced apoptosis, while JNK phosphorylation had a small effect and p38 was not involved. In vivo assay showed that PS inhibited L1210 cell growth in vivo and prolonged the life span of L1210-bearing mice. We conclude that PS is a polysaccharide with anticancer effects and induced apoptosis in human K562 cells.     

Regulation of intracellular iron distribution in K562 human erythroleukemia cells

Following a pulse with 59Fe-transferrin, K562 erythroleukemia cells incorporate a significant amount of 59Fe into ferritin. Conditions or manipulations which alter the supply of iron to cells result in changes in the rate of ferritin biosynthesis with consequent variations in the size of the ferritin pool. Overnight exposure to iron donors such as diferric transferrin or hemin increases the ferritin level 2-4- or 6-8-fold above that of the control, respectively. Treatment with the anti-human transferrin receptor antibody, OKT9 (which reduces the iron uptake by decreasing the number of transferrin receptors) lowers the ferritin level by approximately 70-80% with respect to the control. The fraction of total cell-associated 59Fe (given as a pulse via transferrin) that becomes ferritin bound is proportional to the actual ferritin level and is independent of the instantaneous amount of iron taken up. This has allowed us to establish a curve that correlates different levels of intracellular ferritin with corresponding percentages of incoming iron delivered to ferritin. Iron released from transferrin appears to distribute to ferritin according to a partition function; the entering load going into ferritin is set for a given ferritin level over a wide range of actual amounts of iron delivered.     

Natural killer function in flow cytometry: identification of human lymphoid subsets able to bind to the NK sensitive target K562.

NK cells are a phenotypically, morphologically and functionally heterogeneous population. This has led to the current thought that the non-MHC restricted cytotoxicity is a cellular function that can be associated to different phenotypes. The recognition of the target cell and the conjugate formation is always the first step that eventually leads to the lysis of target. Characterization of the phenotypical pattern of the cells able to bind to K562 targets is the purpose of this study. A multi-parametric flow cytometry binding assay has been employed to identify the different K562-bound lymphocyte subsets. In particular, cells that coexpress the CD16 and CD8 antigens (CD16+8dim+) showed a significantly higher binding capacity than their CD16+8- counterpart. Moreover, the highest binding values have been found in cells that did not express the CD16 antigen at all, but still expressed the CD8dim antigen, such as the small CD8dim+3+ population. These data show that, the NK lytic function being dependent on binding, minor subpopulations must be considered among effector cells, which might correspond to different lytic activities. None of the previously published methodologies that analyze conjugates by flow cytometry or fluorescence microscopy were able to measure the binding capacity of small, double stained, lymphocyte subsets.     

In vitro inhibition of human liver drug metabolizing enzymes by second generation antihistamines

Cetirizine, terfenadine, loratadine, astemizole and mizolastine were compared for their ability to inhibit marker activities for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and for some glucuronidation isoenzymes in human liver microsomes. The most pronounced effects were observed with terfenadine, astemizole and loratadine which inhibited CYP3A4-mediated testosterone 6beta-hydroxylation (IC50 of 23, 21 and 32 microM, respectively) and CYP2D6-mediated dextromethorphan O-demethylation (IC50 of 18, 36 and 15 microM, respectively). In addition, loratadine markedly inhibited the CYP2C19 marker activity, (S)-mephenytoin 4-hydroxylation (Ki of 0.17 microM). Furthermore, loratadine activated the CYP2C9-catalyzed tolbutamide hydroxylation (ca. 3-fold increase at 30 microM) and inhibited some glucuronidation enzymes. Mizolastine appeared to be a relatively weak and unspecific inhibitor of CYP2E1, CYP2C9, CYP2D6 and CYP3A4 (IC50Ss in the 100 micromolar range). Cetirizine demonstrated no effect on the investigated activities. A comparison of the inhibitory potencies of cetirizine, terfenadine, loratidine, astemizole and mizolastine with their corresponding plasma concentrations in humans suggests that these antihistamines are not likely to interfere with the metabolic clearance of coadministered drugs, with the exception of loratidine, which appears to inhibit CYP2C19 with sufficient potency to warrant additional investigation.     

Quantitative morphological analysis of adriamycin-resistant human K562 leukemic cells

The morphological changes associated with Adriamycin resistance in a human leukemic cell line have been investigated by image analysis. An Adriamycin-resistant subline of the human erythroleukemic K562 cell line has been established. Three sets of cells have been analysed: sensitive cells, resistant cells cultured in the continuous presence of Adriamycin, and resistant cells cultured without the drug. Image analysis shows that Adriamycin-resistant K562 cells display significant morphological changes as compared with sensitive cells, at both the nuclear and cytoplasmic levels. These changes make it possible to separate sensitive and resistant cells automatically and with a classification accuracy of 76% and only four cytological parameters. Image analysis may therefore offer an interesting tool for studying drug resistance in leukemic cells, from both an experimental and a clinical point of view.