硼替佐米联合二硫化二砷诱导慢性粒细胞白血病细胞凋亡机制研究

目的:探讨硼替佐米(bortezomib)与二硫化二砷(As2S2)对慢性粒细胞白血病(chronic myeloid leukemia,CML)细胞株K562和对伊马替尼耐药的同源细胞株K562R的增殖抑制、诱导凋亡及其作用机制。方法:应用四甲基偶氮唑盐比色法(MTT)增殖抑制实验、瑞氏染色细胞形态观察法、膜联蛋白Ⅴ(Annexin Ⅴ)流式细胞仪检测硼替佐米和As2S2单独或联合作用于CML细胞株的增殖抑制和凋亡情况;蛋白免疫印迹法(Western blot)检测药物对Bcr-Abl蛋白以及凋亡相关蛋白的影响;反转录PCR(RT-PCR)检测Bcr-Abl mRNA的表达。结果:硼替佐米联合As2S2能有效抑制CML细胞增殖,6nmol/L硼替佐米和3μmol/L As2S2联合作用于K562细胞48h,细胞生长抑制率达(76.4±3.9)%以上,坏死和凋亡细胞比例达54.0%,与单药作用(硼替佐米13.9%;As2S2 8.2%)相比,差异有统计学意义(P<0.05)。药物联合作用通过线粒体途径协同诱导CML细胞凋亡,并使K562和K562R细胞的Bcr-Abl蛋白表达和蛋白磷酸化水平下降。结论:硼替佐米联合As2S2有效抑制K562细胞增殖和诱导凋亡,提高浓度后对K562R细胞也产生类似的作用,有增殖抑制和诱导凋亡作用,两者联合应用可能具有克服伊马替尼耐药的作用。     

Expression of red cell specific determinants during differentiation in the K562 erythroleukaemia cell line

A human haemopoietic cell line (K562) which exhibits various erythroid characteristics, has been utilised as a model system for studying erythropoietic differentiation. We have analysed the alterations in expression of cell membrane determinants which accompany the induction of Hb synthesis. We confirm that the K562 cell line exhibits a number of erythroid features: it expresses immunologically detectable membrane proteins, glycophorin A and spectrin and can ber induced, by addition of haemin or n-butyrate, to synthesise Hb. N and i-like blood group activities are demonstrable in uninduced K562, but band 3, ABH and other major alloantigens characteristic of mature erythrocytes are lacking. The acquisition of cell surface antigens typifying erythroid or other haemopoietic lineages has not been demonstrated following induction, although certain properties not associated with morphologically recognisable red cell precursors are lost. The relevance of these findings to the foetal nature and possible multipotentiality of the K562 cell line are discussed.     

Accumulation of γ-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine

Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo-fetal globin genes, such as the zeta-, epsilon- and gamma-globin genes. The K562 cell line has been proposed as a very useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation stimulating gamma-globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal beta-globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of gamma-globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine-mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of gamma-globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.     

Cyclosporin A induces erythroid differentiation of K562 cells through p38 MAPK and ERK pathways

We studied the effects of cyclosporin A (CsA) on the erythroid differentiation of human erythroid leukemia cell line K562. After K562 was treated with CsA for 4 days, the percentage of hemoglobinized cells was increased by 3.3 times. Because it was reported p38 MAPK (p38) and ERK are involved in erythropoietin-induced erythroid differentiation, we studied their roles using specific inhibitors. p38 inhibitor (SB203580) prevented CsA-induced hemoglobin synthesis in K562 cells, although MEK/ERK inhibitor (U0126) enhanced it by 3.3 times in K562 cells. These results indicate activation of p38 and inactivation of ERK are involved in CsA-induced erythroid differentiation of K562 cells.     

Increase in γ-globin mRNA content in human erythroid cells treated with angelicin analogs

The aim of the present study was to identify molecular analogs of angelicin (ANG) able to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells, with low effects on apoptosis. ANG-like molecules are well-known photosensitizers largely used for their antiproliferative activity in the treatment of different skin diseases (i.e., psoriasis, vitiligo, eczema, and mycosis fungoides). To verify the activity of these derivatives, we employed three experimental cell systems: (1) the human leukemic K562 cell line, (2) K562 cell clones stably transfected with a pCCL construct carrying green-EGFP under the γ-globin gene promoter, and (3) the two-phase liquid culture of human erythroid progenitors isolated from normal donors and β-thalassemia patients. The results of our study suggest that trimethyl ANG is a powerful inducer of erythroid differentiation, compared with known inducers, such as ANG, cytosine arabinoside, mithramycin, and cisplatin. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered a potential therapeutic approach in hematological disorders including β-thalassemia and sickle cell anemia.     

Butyrate-induced erythroid differentiation of human K562 leukemia cells involves inhibition of ERK and activation of p38 MAP kinase pathways

Butyrate induces cytodifferentiation in many tumor cells of different origin, suggesting that an as yet unidentified common mechanism inherent to malignant cells is the target of butyrate action. This study determined the role of different mitogen-activated protein (MAP) kinase signal transduction pathways in butyrate-induced erythroid differentiation of K562 human leukemia cells. Using a panel of anti-ERK, JNK, and p38 phosphospecific antibodies, the study showed that phosphorylation of ERK and JNK is decreased following treatment of cells with butyrate, whereas phosphorylation of p38 is increased. In contrast, a K562 subline defective in butyrate-mediated induction of erythroid differentiation did not reveal these changes in phosphorylation patterns. Inhibition of ERK activity by UO126 induces erythroid differentiation and acts synergistically with butyrate on hemoglobin synthesis and inhibition of cell proliferation, whereas inhibition of p38 activity by SB203580 completely abolished induction of hemoglobin expression by butyrate. Taken together, our data suggest a model in which butyrate induces erythroid differentiation of K562 cells by inhibition of ERK and activation of p38 signal transduction pathways.     

A monoclonal antibody that detects expression of transferrin receptor in human erythroid precursor cells

A monoclonal antibody, L5.1, obtained by immunizing a Balb/c mouse with HL60 human promyelocytic leukemia cells, was found to react with both HL60 cells and with the K562(S) cell line. This monoclonal antibody binds and immunoprecipitates a glycoprotein (Mr 87,000) present on the cell surface membrane of K562(S) as a disulfide bonded dimer. In competition experiments L5.1 competes with both transferrin and OKT9 (a known antitransferrin receptor antibody) for binding to target K562(S) erythroleukemia cells. Binding of both L5.1 and transferrin to the surface of K562(S) cells is inhibited by treatment with 12--O- tetradecanoyl-phorbol-13-acetate, and the extent and time course of inhibition is similar in both cases. Cell sorting analysis of normal human marrow cells incubated with L5.1 indicates that L5.1 reacts strongly with all the morphologically recognizable erythroid lineage precursors, from the pronormoblast to the orthochromatic normoblast, and with reticulocytes. Erythrocytes, myeloid elements, monocytes, megakaryocytes and platelets, peripheral blood B and T lymphocytes do not bind significantly with this antibody and only a small fraction of promyelocytes was reactive. Antibody L5.1 did not react with leukemic cells of patients with acute lymphoblastic, myeloblastic and promyelocytic leukemias, but it did react with some established B (1 of 5) and T (2 of 3) cell lines, and a myeloid (1 of 3) cell line, and with PHA-stimulated peripheral blood lymphocytes. The nonhemopoietic cell lines tested did not bind with L5.1 with the exception of a colorectal adenocarcinoma and a melanoma cell line, which were both strongly positive. The relationship of antibody L5.1 to other monoclonal antibodies that bind the transferrin receptor is discussed.     

Induction of erythropoietic colonies in a human chronic myelogenous leukemia cell line.

The ability of cells derived from the K562 cell line to generate erythropoietic colonies was studied. The K562 cell line was derived from a patient with chronic myelogenous leukemia 8 yr ago by Lozzio and Lozzio. Rare benzidine-positive colonies formed when these cells were cloned in plasma clots (3 +/- 1/10(4) cells), and their number was not substantially increased by the addition of erythropoietin (9.5 +/- 1/10(4) cells). Sodium butyrate was capable of markedly enhancing the number of benzidine-positive colonies (19.5 +/- 1/10(4) cells) formed, while the combination of sodium butyrate plus erythropoietin exerted a synergistic effect on erythropoietic colony formation (57 +/- 4/10(4) cells). The K562 cell line is a long-term culture system that contains human erythropoietic stem cells. This cell line should be useful in future studies on the cellular and molecular events associated with human erythroid cell differentiation.     

Enhanced expression of transforming growth factor beta during megakaryoblastic differentiation of K562 leukemia cells

Platelet alpha granules contain several growth factors such as the transforming growth factor beta (TGF-beta) that are released during blood clotting and are thought to participate in the repair of tissue injury; however, the site of synthesis of platelet TGF-beta has not been demonstrated. We studied TGF-beta expression during megakaryoblastic differentiation of the chronic myeloid leukemia cell line K562 in vitro. These cells have mainly erythroid characteristics but acquire several megakaryoblastic properties when treated with the phorbol diester 12-0-tetradecanoyl-13-phorbolacetate (TPA). During four subsequent days of megakaryoblastic differentiation the amount of the 2.5-kilobase (kb) TGF-beta mRNA increased about eightfold, and a novel 2.3-kb mRNA species was induced in the K562 cells. This occurred concomitantly with distinct induction patterns of platelet-derived growth factor A (PDGF-A) and c-sis (PDGF-B chain) RNAs and several platelet antigens. The expression of erythroid markers such as glycophorin A decreased. Culture media of TPA-differentiated K562 cells also contained TGF-beta polypeptides as shown by a sensitive radioreceptor assay and by immunoprecipitation after metabolic labeling of the cells. These polypeptides were not seen in culture media from dimethyl sulfoxide- or sodium butyrate-treated cells. Unlike in several other cells, exogenously added TGF-beta 1 or 2 affected neither TGF- beta nor PDGF RNA expression in K562 cells.     

Expression of interleukin 6 and its specific receptor by untreated and PMA-stimulated human erythroid and megakaryocytic cell lines

Different human hematopoietic cell lines were analyzed for the presence of interleukin 6 (IL-6) and IL-6 receptor (IL-6-R). Both IL-6 mRNA and secreted active IL-6 protein were detectable in untreated cell lines with erythroid or megakaryoblastic features (K562, HEL, KU 812, MEG-01, and Dami), but they were not expressed constitutively in other leukemic cell lines (KG1, HL60, and U937). IL-6-R production, studied by the presence of its mRNA and specific binding sites for iodinated recombinant IL-6, was detected in most cell lines except K562, HEL, and Dami. Therefore, only KU 812 and MEG-01 coexpress both IL-6 and IL-6-R. After phorbol ester myristate acetate (PMA) treatment, all the cell lines studied expressed or overexpressed IL-6. In the erythroid K562 cell line, IL-6-R was not detectable before induction, but was promptly expressed after stimulation with PMA, suggesting that some of the new features of K562 cells induced by PMA may be mediated by IL-6. However, neutralizing antibodies against IL-6 did not block either the growth arrest or the loss of the erythroid phenotype induced by PMA. The presence of IL-6 and IL-6-R in erythroid and megakaryocytic leukemic cell lines suggests that their synthesis may occur during normal hematopoietic differentiation.     

Inhibition of hemoglobin accumulation by monoclonal antibodies to the human transferrin receptor is reversed by hemin

We have studied the effects of hemin on the inhibition of K562 cell erythroid differentiation mediated by a monoclonal antibody to the human transferrin receptor. In the absence of hemin, the monoclonal antibody 42/6 suppresses the increase of hemoglobin accumulation induced by butyric acid or 5-azacytidine (5-azaCR). In contrast, in the presence of hemin, the hemin-resistant K562(h) cell line is induced to hemoglobin production by treatment with 5-azaCR even in the presence of the monoclonal antibody to human transferrin receptor. The results obtained suggest (1) that the human leukemic K562 cells treated with monoclonal antibodies to human transferrin receptor might retain molecular properties similar to those found in erythroid cells from subjects affected by alterations of iron metabolism, and (2) that hemin might allow hemoglobin synthesis in erythroid cells whose iron uptake is inhibited by monoclonal antibodies to human transferrin receptors.     

Accumulation of gamma-globin mRNA in human erythroid cells treated with angelicin

The aim of the present study was to determine whether angelicin is able to increase the expression of gamma-globin genes in human erythroid cells. Angelicin is structurally related to psoralens, a well-known chemical class of photosensitizers used for their antiproliferative activity in treatment of different skin diseases (i.e., psoriasis and vitiligo). To verify the activity of angelicin, we employed two experimental cell systems, the human leukemic K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors. The results of our investigation suggest that angelicin, compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and gamma-globin mRNA accumulation of human leukemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of angelicin, increases of gamma-globin mRNA accumulation and fetal hemoglobin (HbF) production, even higher than those obtained using hydroxyurea, were detected. These results could have practical relevance, as pharmacologically-mediated regulation of the expression of human gamma-globin genes, leading to HbF induction, is considered a potential therapeutic approach in hematological disorders, including beta-thalassemia and sickle cell anemia.     

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the zeta, epsilon and gamma globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper we demonstrated that the G + C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) gamma-globin mRNA.     

Apoptosis induced by erythroid differentiation of human leukemia cell lines is inhibited by Bcl-XL

The induction of tumor cell differentiation represents an attractive strategy for the treatment of a wide range of malignancies. Differentiation of HL-60 promyelocytic leukemia cells towards neutrophils or monocytes has been shown to induce apoptotic cell death, which is inhibited by bcl-2 over-expression. However, the role of the bcl-2 gene family during erythroid differentiation of human leukemia cells remains unknown. We found that human erythroleukemia (HEL) and K562, two leukemia cell lines that undergo erythroid differentiation do not express Bcl-2, but express Bcl-XL, a related protein that functions as an inhibitor of apoptosis. Differentiation of HEL or K562 cells with inducers of erythroid differentiation (hemin, retinoic acid, or transforming growth factor-beta) was accompanied by progressive cell death and degradation of genomic DNA into oligonucleosomal fragments. The loss of cellular viability was associated with downregulation of bcl-xL mRNA and protein. In contrast, the levels of Bax, another Bcl-2 family member implicated in apoptosis remained unaltered. Constitutive expression of Bcl-XL by gene transfer inhibited apoptosis triggered by erythroid differentiation of HEL K562 cells. Yet, Bcl-XL did not alter the expression of epsilon-globin, which is induced during erythoid differentiation of HEL and K562 cells, arguing that apoptosis and differentiation can be uncoupled by Bcl-XL. These results indicate that Bcl-XL acts as an antiapoptosis protein in leukemia cells that undergo erythroid differentiation and that downregulation of bcl-x is a component of the apoptotic response that is coupled to differentiation in human leukemia cells.     

Cyclic changes in fluorescence polarization of a membrane probe during the cell cycle of an erythroid precursor cell line

The membrane lipid bilayer of the K562 cell line undergoes marked changes during cell cycling. These changes can be detected by measuring the fluorescence polarization of the rod-like hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene. Single-cell measurements were performed by flow microfluorometry on synchronized K562 cells. The fluorescence polarization of the probe increased after cell division and was maximal during the S phase. Concomitant with the transition from S to G2 and M phase was a decrease in fluorescence polarization. The data are interpreted as reflecting a minimal membrane lipid fluidity during the S phase and a maximal fluidity during the G2/M-phase of the above erythroid precursor cell line.