Alternate iron transport pathway. Mobilferrin and integrin in K562 cells

A transferrin-independent iron transport system in cells containing transferrin receptors was described previously by several investigators. Prior studies did not identify the proteins involved in this alternate iron transport pathway. Using a human-derived erythroleukemia tissue culture line, iron-binding proteins were isolated from cytosol and cell membranes. The cytosol protein was soluble in 60% ammonium sulfate, had a molecular mass similar to mobilferrin (56 kDa), and reacted with anti-mobilferrin antibodies. The water-insoluble radiolabeled protein was solubilized with Nonidet P-40 and immunoprecipitated with monoclonal antibody against beta 3 human integrin. Pulse-chase studies suggested sequential passage of iron to integrin, mobilferrin, and ferritin, respectively. Thus, the alternate iron transport pathway contained proteins similar to those observed in intestinal cells which did not possess transferrin receptors on their absorptive surface. The alternate iron transport pathway is only partially shared with zinc and cadmium. Mobilferrin bound zinc and iron competitively, but the two metals were not transported competitively into K562 cells. Immunoprecipitates of integrin containing radiozinc were obtained with a monoclonal antibody against beta 1 human integrin. This suggested iron and zinc may utilize different integrins to passage the cell membrane.     

Enforced expression of Bcl-XS induces differentiation and sensitizes chronic myelogenous leukemia-blast crisis K562 cells to 1-beta-D-arabinofuranosylcytosine-mediated differentiation and apoptosis

Human chronic myelogenous leukemia-blast crisis K562 cells have been demonstrated to be relatively resistant to antileukemic drug-induced apoptosis. This has been attributed to the activity of p210bcr-abl tyrosine kinase present in the K562 cells, which is known to suppress drug-induced apoptosis. Recently, K562 cells have been shown to express the antiapoptosis Bcl-xL but not Bcl-2 proteins. To investigate the contribution of Bcl-xL toward resistance to drug-induced apoptosis, we created K562/Bcl-xS and K562/neo cells by electroporating the expression plasmids pSFFVneo-Bcl-xS and pSFFVneo, containing the bcl-xS and neomycin resistance genes, respectively, into K562 cells. K562/Bcl-xS but not K562/neo cells expressed the bcl-xS mRNA and p19Bcl-xS protein. In contrast, both cell types expressed equivalent levels of Bcl-xL, Bax, Bcl-2, Myc, retinoblastoma, p21cbor-abl, and p145abl proteins. A significant increase in the hemoglobin levels was observed in the K562/Bcl-xS compared with the K562/neo cells (P < 0.05). In addition, K562/Bcl-xS cells were significantly more sensitive than K562/neo cells to undergoing erythroid differentiation induced by low-dose 1-beta-D-arabinofuranosylcytosine (ara-C) and hexamethyl bisacetamide (P < 0.05), but not by all-trans-retinoic acid. Low-dose ara-C- or hexamethyl bisacetamide-induced differentiation was not associated with apoptosis of K562/Bcl-xS or K562/neo cells. Low-dose ara-C-induced erythroid differentiation was accompanied by conversion of the retinoblastoma protein to predominantly its underphosphorylated isoform as well as by down-regulation of Myc levels in K562/Bcl-xS and K562/neo cells. Importantly, exposure to high-dose ara-C (HIDAC; 100 microM ara-C for 4 h) caused internucleosomal DNA fragmentation and the morphological features of apoptosis in K562/Bcl-xS cells. These effects were modestly enhanced by cotreatment with HIDAC plus herbimycin A. In contrast, K562/neo cells were completely resistant to HIDAC- and herbimycin A-induced apoptosis. These results indicate that the expression of Bcl-xS induces erythroid differentiation and partially sensitizes chronic myelogenous leukemia-blast crisis-derived K562 cells to ara-C-induced differentiation and apoptosis.     

Upregulation of CD9 expression during TPA treatment of K562 cells

The CD9 antigen, a major platelet glycoprotein, is a member of the tetraspan superfamily. We show that treatment of K562 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) which induces megakaryocytic differentiation, leads to a seven-fold increase in CD9 expression, which becomes associated with the integrin beta1, suggesting that it is functionally relevant. The upregulation of CD9 expression precedes the appearance of the megakaryocytic-specific marker GPIIb (CD41) as well as integrins beta3 (GPIIIa/CD61), alpha v (CD51) and VLA-2 (CD49b). Both GPIIb/IIIa expression and CD9 upregulation are dependent on protein kinase C (PKC) activation since they are blocked by the specific inhibitor GF109203X. Steady-state levels of CD9 and GPIIb mRNA were also measured by quantitative RT-PCR. Both messengers were detected on resting cells and were shown to accumulate during TPA treatment. However, the increase of the CD9 mRNA was detected much earlier than the increase of GPIIb mRNA (1-2 h vs 24-48 h). Using different constructs of the 5'-flanking domain of the CD9 gene cloned ahead of the CAT reporter gene, we could demonstrate that a responsive element was located in a 52 bp fragment of the promoter of the CD9 gene. Altogether, these data suggest that CD9 upregulation in the megakaryocytic lineage could occur at early stages of differentiation.     

The dendritic cell differentiation and antigen-presenting function of the erythroleukemia cells induced by GM-CSF

OBJECTIVE: To investigate the generation of dendritic cells and the change of antigen-presenting function during the differentiation of FBL-3 erythroleukemia cells induced by GM-CSF. METHODS: The effects of GM-CSF on the phenotype, ultrastructure and antigen-presenting function of FBL-3 erythroleukemia cells were observed by FACS, electromicroscopy and 51Cr-release assay. RESULTS: After treatment with 100 ng/ml GM-CSF for 3 days, the expressions of 33 D1 and NLDC 145 which are the specific markers on dendritic cells were increased significantly; MHC-II B7-1, B7-2, ICAM-1, VCAM-1 and CD40 were also upregulated. The membrane of FBL-3 cells was changed into villous surface with dendritic projections. There were plenty of mitochondria in cytoplasm, the nucleus became lobulated. The GM-CSF-treated FBL-3 cells could apparently stimulate the proliferation of allogeneic T lymphocytes, induce the production of IL-2 and improve the specific cytotoxic activity of CTL on FBL-3 cells. CONCLUSION: Erythroleukemia cells were induced to differentiate into the dendritic cells by GM-CSF and obtained the antigen-presenting function.     

K562细胞硫氧还蛋白还原酶活力测定及其抑制剂体外抗白血病的初步研究

目的 探讨慢性粒细胞白血病(CML)细胞株K562中硫氧还蛋白还原酶(TrxR)的活力及其新型抑制剂乙烷硒啉(BBSKE)体外抗白血病作用.方法 应用胰岛素还原法检测K562细胞株及健康人骨髓单个核细胞中TrxR的活力.运用CCK-8法测定BBSKE对K562细胞的增殖抑制率.应用激光共聚焦显微镜、琼脂糖凝胶电泳以及Annexin V-FITC/PI双标记流式细胞术观察BBSKE的抗白血病作用.结果 K562细胞中TrxR的活性明显高于健康人骨髓单个核细胞,10 μmol/L BBSKE与K562细胞作用24 h,激光共聚焦显微镜可见典型的细胞凋亡表现,琼脂糖凝胶电泳后可见典型的DNA"梯"条带出现,流式细胞术检测凋亡率为(10.28±2.74)%;10 μmol/LBBSKE对CML患者原代细胞有诱导凋亡的作用,凋亡率为(5.70±0.48)%.结论 慢性粒细胞白血病细胞株K562中TrxR活力高于健康人骨髓单个核细胞,BBSKE有抑制TrxR活力、抑制K562细胞增殖和诱导凋亡的作用,是治疗CML潜在的有效药物.     

Terminal differentiation of murine erythroleukemia cells: physical stabilization of end-stage cells

An important limitation in the use of the murine erythroleukenia (MEL) cell system as an in vitro system for the study of terminal erythroid differentiation has been the inability to produce significant numbers of cells which represent the end-point of the pathway in vitro. We show here that a major reason for the failure to observe end-stage cells in vitro is that such cells are physically unstable under the standard culture conditions used for MEL cell differentiation. Modification of these culture conditions by the addition of either bovine serum albumin or Ficoll leads to physical stabilization of end-stage cells. Under such culture conditions, uniform cultures of terminally differentiated MEL cells with morphological characteristics similar to those of normal mouse orthochromatophilic erythroblasts and reticulocytes are observed. Examination of physical and biochemical parameters of these cell populations give values which are similar to values characteristic of mouse reticulocytes. A physically stabilized MEL cell shows a narrow cell volume distribution with an average value of approximately 100 mum(3), similar to the cell volume distribution observed for mouse reticulocytes, while a typical MEL cell culture treated with DMSO but without a stabilizing agent exhibits a broader, more heterogeneous cell volume distribution with an average value of approximately 500 mum(3). Globin mRNA levels and levels of globin synthesis reach values almost equal to those in mouse reticulocytes in cultures of physically stabilized MEL cells while differentiating cultures not treated with a stabilizing agent reach substantially lower values for these parameters. We suggest that the ability to produce populations of MEL cells which undergo complete terminal erythroid differentiation in vitro will allow the analysis of the molecular mechanisms which control the terminal stages of the erythroid differentiation process.     

Wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, inhibits erythropoietin-induced erythroid differentiation of K562 cells

We report a large kindred in which a punctate palmoplantar keratoderma (PPK) is associated with malignancy, including Hodgkin's disease, renal, breast, pancreatic and colonic adenocarcinomas. The family was traced through four generations, and over 320 individuals were identified, of whom 49 had punctate PPK. The punctate PPK appeared to be inherited as an autosomal dominant trait with variable penetrance. Ten of the 43 adults (23%) with punctate PPK developed malignancies, and five of these developed before the age of 50. Of the 271 unaffected individuals, six (2%) have developed malignancies, one prior to the age of 50. The association of keratoderma and malignancy is discussed.     

Sensitivity of vincristine-sensitive K562 and vincristine-resistant K562-Lucena 1 cells to anthracyclines and reversal of multidrug resistance

The phenomenon of multidrug resistance (MDR), that involves the efflux pump P-glycoprotein, can be reversed by a number of substances known as MDR modulators or reversing agents. In the present study we investigated the action of three anthracyclines, mitoxantrone and vincristine on short-term (72 h) cultures using 2 methods ([3H] incorporation and MTT (3-[4,5-dimethylthiasol-2-yl]-2,5-diphenyltetrazolium bromide)), on 2 cell lines: K562, a human erythroleukemia, and a vincristine-resistant subline K562-Lucena 1. Using the same culture methods plus flow cytometry analysis, the reversing potentials of cyclosporin A and verapamil were studied in both cell lines. There were differences in the sensitivity and resistance profiles of the two lines to the various drugs but daunorubicin (5 micrograms/ml) and idarubicin (0.035 micrograms/ml) were the most effective when each was used in high concentration. Cyclosporine at 200 ng/ml and verapamil at 5 micrograms/ml reversed MDR in the resistant line, and had a synergistic action with chemotherapeutic agents on the sensitive line. Again differences were demonstrable between combinations of the various drugs and reversal was only clearly shown with the method measuring cell proliferation ([3H] incorporation) but not by the method measuring metabolic activity (MTT). The efflux of rhodamine-123 mimics the functional activity of the pump and cyclosporine was a better reversing agent by this criteria. These data show that the results obtained in in vitro studies attempting to identify treatments for different types of leukemias depend to a large extent on the methods used to measure cell response.     

Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells

Binding experiments with the specific muscarinic ligand [3H]N-methylscopolamine (3H-NMS) have shown the presence of constitutive muscarinic acetylcholine receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1-M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K(D)) for 3H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B+) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B+ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells.     

Trimidox-mediated morphological changes during erythroid differentiation is associated with the stimulation of hemoglobin and F-cell production in human K562 cells

Previous studies have demonstrated that stimulation of the ventral hippocampal (VH) formation (including the ventral CA1 and subicular areas) elicits increased locomotor activity in rats. The locomotor-activating effects of VH stimulation have been hypothesized to be mediated via hippocampal output to cortical and subcortical dopamine (DA) systems. This study examined whether increased locomotor activity produced by VH stimulation was blocked by pretreatment with a DA receptor antagonist, and whether DA metabolism in subdivisions of the nucleus accumbens, caudate-putamen, and prefrontal cortex was elevated by VH stimulation. Stimulation of the VH (defined as the ventral CA1 and its borders, ventral subiculum, and entorhinal cortex) with the cholinergic agonist carbachol was found to elevate locomotor activity, while pretreatment with the D2 receptor antagonist haloperidol blocked this effect. Stimulation of the VH did not alter DA metabolism (i.e., ratio of the DA metabolites DOPAC or HVA/DA) in any of the brain regions studied. These results indicate that the increased locomotor activity elicited by VH stimulation is not associated with dramatic increases in DA metabolism, but that it does require tonic activation of D2 receptors.     

Protein kinase C isotypes in human erythroleukemia (K562) cell proliferation and differentiation. Evidence that beta II protein kinase C is required for proliferation

The human erythroleukemia (K562) cell line undergoes megakaryocytic differentiation and cessation of proliferation when treated with phorbol myristate acetate (PMA). To investigate the role of individual protein kinase C (PKC) isotypes in these events, we have assessed PKC isotype expression during leukemic proliferation and PMA-induced differentiation. Immunoblot analysis using isotype-specific antibodies demonstrates that proliferating K562 cells express the alpha, beta II, and zeta PKC isotypes. PMA-induced differentiation and cytostasis lead to a decrease in beta II PKC and increases in alpha and zeta PKC levels. The role of the alpha and beta II PKC isotypes was further assessed in cells overexpressing these isotypes. K562 cells overexpressing human alpha PKC grew more slowly and were more sensitive to the cytostatic effects of PMA than control cells, whereas cells overexpressing beta II PKC were less sensitive to PMA. PMA-induced cytostasis is reversed upon removal of PMA. Resumption of proliferation is accompanied by reexpression of beta II PKC to near control levels, whereas alpha and zeta PKC levels remain elevated for several days after removal of PMA. Proliferation of PMA-withdrawn cells can be partially inhibited by antisense beta II PKC oligodeoxyribonucleotide. Growth inhibition is dose-dependent, specific for beta II PKC-directed antisense oligonucleotide, and associated with significant inhibition of beta II PKC levels indicating that beta II PKC is essential for K562 cell proliferation. Sodium butyrate, which unlike PMA induces megakaryocytic differentiation without cytostasis, causes increases in both alpha and beta II PKC levels. These data demonstrate that beta II PKC is required for K562 cell proliferation, whereas alpha PKC is involved in megakaryocytic differentiation.     

Erythroid differentiation and growth inhibition of K562 cells by 2',5'-dideoxyadenosine: synergism with interferon-alpha

We found that 2',5'-dideoxyadenosine (DDA), a P-site specific adenylate cyclase inhibitor, inhibited the growth of K562 cells and caused them to become benzidine positive. The continuous exposure of cells to DDA was needed to recruit cells for growth inhibition and differentiation. Fetal calf or human sera were also necessary for DDA to induce differentiation. DDA at a concentration of 1.5 mM with serum induced 98% of the cells to produce hemoglobin and inhibited their growth to 15% of that of the control. An increase of epsilon-globin mRNA and a decrease of c-myc and c-myb mRNA occurred only during differentiation in the presence of fetal calf serum (FCS). An incubation with DDA and interferon-alpha (IFN-alpha) or hemin synergistically induced more benzidine-positive cells than in the presence of DDA alone, although IFN-alpha did not trigger differentiation by itself. The erythroid differentiation and growth inhibition were, however, not related to a decreased intracellular cyclic AMP (cAMP) concentration induced by DDA. The simultaneous incubation with dibutyryl cyclic AMP (dbc-AMP) and DDA enhanced the effects of DDA. Adenine, a possible metabolite of DDA digestion by purine nucleoside phosphorylase (PNP), also induced erythroid differentiation in K562 cells. However, it did not act synergistically with IFN-alpha.     

Nuclear but not cytoplasmic phospholipase C beta 1 inhibits differentiation of erythroleukemia cells

A body of evidence has shown the existence of a nuclear phosphoinositide cycle in different cell types. The cycle is endowed with kinases as well as phosphatases and phospholipase C (PLC). Among the PLC isozymes, the beta family is characterized by a long COOH-terminal tail that contains a cluster of lysine residues responsible for nuclear localization. Indeed, PLC beta 1 is the major isoform that has been detected in the nucleus of several cells. This isoform is activated by insulin-like growth factor I, and when this isoform is lacking, as a result of gene ablation, the onset of DNA synthesis induced by this hormone is abolished. On the contrary, PLC beta 1 is down-regulated during the erythroid differentiation of Friend erythroleukemia cells. A key question is how PLC beta 1 signaling at the nucleus fits into the erythroid differentiation program of Friend erythroleukemia cells, and whether PLC beta 1 signaling activity is directly responsible for the maintenance of the undifferentiated state of erythroleukemia cells. Here we present evidence that nuclear PLC beta 1 but not the isoform located at the plasma membrane is directly involved in maintaining the undifferentiated state of Friend erythroleukemia cells. Indeed, when wild-type PLC beta 1 is overexpressed in these cells, differentiation in response to DMSO is inhibited in that the expression of beta-globin is almost completely abolished, whereas when a mutant lacking the ability to localize to the nucleus is expressed, the cells differentiate, and the expression of beta-globin is the same as in wild-type cells.     

Induction of apoptosis in human leukemia K562 cells by alpha-anordrin

The apparent coexistence of primary biliary cirrhosis (PBC) and autoimmune hepatitis in the same patient raises unresolved problems for nosology and therapy. These are exemplified by a 45-year-old Japanese woman with overlapping clinical, serological and histological features of autoimmune cholangitis and autoimmune hepatitis. The classical serological test for PBC, antimitochondrial antibody (AMA) by immunofluorescence, was atypical. By immunoblotting there was reactivity with one of the enzymes of the 2-oxo-acid dehydrogenase complex (2-OADC) family, now recognized as autoantigens responsible for AMA reactivity. Also there was reactivity by immunofluorescence for antinuclear antibodies (ANA), one showing the typical speckled pattern of anti-Sp-100 and the other the peripheral pattern of antinuclear membrane antibody, both with titres > 10(6). There was also a positive result to the lupus erythematosus (LE) cell test. Treatment with ursodeoxycholic acid was beneficial. Thus while the clinical presentation suggested the overlapping syndrome of autoimmune hepatitis and PBC, PBC eventually proved to be the likely diagnosis. We suggest that apparent cases of overlapping PBC-autoimmune cholangitis-hepatitis syndromes, after detailed testing, will mostly align with PBC.     

Protein tyrosine phosphatase PTP1B suppresses p210 bcr-abl-induced transformation of rat-1 fibroblasts and promotes differentiation of K562 cells

The bcr-abl chimeric oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias, such as chronic myelogenous leukemia (CML). Recently we have shown that the levels of the protein tyrosine phosphatase PTP1B are enhanced in p210 bcr-abl-expressing cell lines. Furthermore, PTP1B recognizes p210 bcr-abl as a substrate, disrupts the formation of a p210 bcr-abl/Grb2 complex, and inhibits signaling events initiated by this oncoprotein PTK. In this report, we have examined whether PTP1B effects transformation induced by p210 bcr-abl. We demonstrate that expression of either wild-type PTP1B or the substrate-trapping mutant form of the enzyme (PTP1B-D181A) in p210 bcr-abl-transformed Rat-1 fibroblasts diminished the ability of these cells to form colonies in soft agar, to grow in reduced serum, and to form tumors in nude mice. In contrast, TCPTP, the closest relative of PTP1B, did not effect p210 bcr-abl-induced transformation. Furthermore, neither PTP1B nor TCPTP inhibited transformation induced by v-Abl. In addition, overexpression of PTP1B or treatment with CGP57148, a small molecule inhibitor of p210 bcr-abl, induced erythroid differentiation of K562 cells, a CML cell line derived from a patient in blast crisis. These data suggest that PTP1B is a selective, endogenous inhibitor of p210 bcr-abl and is likely to be important in the pathogenesis of CML.