Constitutive expression of platelet glycoproteins by the human leukemia cell line K562

The human leukemia cell line K562 was derived from a patient with chronic granulocytic leukemia. This cell line has subsequently been shown to possess phenotypic markers typical of erythroid and myeloid cells. Using a rabbit antiserum directed against purified platelet glycoproteins (PGPs), we have obtained evidence for the constitutive expression of PGPs on the surface of K562 cells. PGPs expressed have been tentatively identified as IIa and III based on their apparent migration in a 7% sodium dodecylsulfate polyacrylamide gel. K562 may become an important tool for the study of early events involved in megakaryocytic differentiation.     

Monoclonal antibodies against the human leukemia cell line K 562

Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.     

运用siRNA抑制K562细胞中端粒酶逆转录酶基因表达的研究

目的建立利用小分子干扰RNA(siRNA)技术抑制白血病细胞系K562细胞中端粒酶活性的方法,为肿瘤的基因治疗提供理论依据。方法设计端粒酶逆转录酶(hTERT)基因特异性siRNA,用体外转录方法合成hTERT基因的siRNA并转染K562细胞,培养48小时后,收集细胞,应用实时荧光定量RT-PCR和western blot方法检测转染细胞中hTERT基因mRNA水平和蛋白表达量的变化,并运用TRAP ELISA方法检测细胞内端粒酶活性的变化。结果转染siRNA后,与对照组相比,实验组hTERT mRNA水平和蛋白表达量明显降低,抑制率分别为75％和60％,同时。TRAP ELISA方法检测发现实验组端粒酶活性仅为对照组活性的45％。结论hTERT siRNA能特异性的抑制hTERT基因的表达,降低端粒酶活性,因此运用siRNA来抑制hTERT的表达可达到降低端粒酶活性的效果。     

Human leukemia cell line K562 responds to erythroid-potentiating activity

We report that erythroid-potentiating activity (EPA), known to stimulate the proliferation of normal human erythroid precursors in vitro, has a growth-promoting effect on human K562 erythroleukemia cells and Friend mouse erythroleukemia cells. Detailed studies were carried out using an EPA produced by a human T-lymphoblast line (Mo). Although EPA has not been purified to homogeneity, several observations indicate that the factor elaborated by Mo cells that stimulates erythroleukemia cell growth is the EPA molecule. The erythroleukemia growth factor cofractionates with EPA using gel exclusion chromatography, isoelectric focusing, and ion exchange chromatography. In addition, the activities exhibit similar kinetics of heat inactivation. A granulocyte-macrophage colony-stimulating factor also elaborated by Mo cells had no effect on the growth of the erythroleukemia cells. Other sources of EPA, such as peripheral blood leukocyte-conditioned medium, preparations from urine of anemic patients, and medium conditioned by a human monocyte-like cell line, stimulated erythroleukemia cell growth. Mouse sources of EPA (termed "burst-promoting activity") stimulated mouse but not human erythroleukemia cells. The availability of cell lines apparently responsive to EPA should prove useful for examining the mode of action of this regulator of erythropoiesis.     

Smad7在人慢性髓细胞白血病细胞株K562中的表达

目的：观察人慢性髓细胞白血病细胞株K562中Smad7基因和蛋白的表达；探讨转化生长因子(TGF—β1）mad7的诱导作用。方法：采用逆转录-聚合酶链反应、蛋白印迹法方法检测K562细胞与TGF-1共育前后Smad7 mRNA蛋白表达水平。结果：K562细胞中具有Smad7的表达，且可由外源性TGF-β1短暂诱导，Smad7的表达及变化在转录及翻译水平上基本保持一致。结论：Smad7参与K562细胞TGF-β信号的传导，Smad7与TGF-β1之间存在自身调节负反馈通路。     

The role of folates in the development of methotrexate resistance in human leukemia cell line K562

The effect of reduced and oxidized folates on the development of methotrexate (MTX) resistance has been examined in human leukemia cell line K562 (K562/S). K562/S cells were made resistant to MTX by soft-agar cloning either in RPMI-1640 medium (K562/MTX-PGA) or in folic-acid-free RPMI-1640 medium containing 10 nM leucovorin (K562/MTX-LV). The optimal concentrations of leucovorin for the growth of K562/S, K562/MTX-PGA and K562/MTX-LV cells were 1 nM, 5 nM and 10 nM respectively. K562/MTX-PGA cells were 24-fold resistant to MTX as noted by impaired MTX transport. In contrast, K562/MTX-LV cells were 26-fold resistant to MTX as noted by gene amplification of dihydrofolate reductase. Furthermore cross-resistance to cytosine arabinoside was only demonstrated in K562/MTX-PGA, while the K562/MTX-LV cells showed no significant cross-resistance to cytosine arabinoside. These results suggest that the type and level of folates used during the development of MTX resistance may play a role in the mechanism for MTX resistance. Leukemia cells that are grown in leucovorin might serve as a model for acquired MTX resistance in vivo.     

Embryonic erythroid differentiation in the human leukemic cell line K562.

K562 human leukemia cells synthesize embryonic hemoglobins after culture in the presence of hemin. We have rigorously identified these hemoglobins by globin chain analysis and peptide mapping. No adult hemoglobin could be detected, and beta-globin synthesis was less than 2 ppm of total protein synthesis. Persistent embryonic globin gene expression is known to occur as a consequence of globin gene deletions. However, restriction endonuclease mapping showed that the globin gene complexes in K562 cells are indistinguishable from normal. Hemin increased the rate of embryonic globin synthesis. The pattern of hemoglobin synthesis proved to be stable when cells from different laboratories were compared. One line, however, synthesized large amounts of Hb X and very little Hb Portland in response to hemin. Hb X has been previously detected in human embryos; we show here that it has the composition epsilon 2 gamma 2 and is diagnostic of imbalanced chain synthesis or "zeta thalassemia." We have identified several agents that induce hemoglobin synthesis in K562 cells. Different inducers induced different patterns of embryonic hemoglobin synthesis but never any adult hemoglobin synthesis.     

齐墩果酸对K562细胞系VEGF表达影响的实验研究

目的观察齐墩果酸(Oleanolic Acid,OA)对慢性粒细胞白血病系K562细胞中血管内皮生长因子(VEGF)表达的影响。方法采用MTT实验观察OA对体外培养K562细胞增殖抑制作用,用RT-PCR以及Western印迹法研究OA处理后K562细胞中VEGF基因表达的变化。并用ELISA方法检测培养液中VEGF的表达。结果OA能抑制K562细胞生长,呈一定的量效特征;并能抑制K562细胞中VEGF的表达。结论OA通过下调VEGF表达,抑制白血病细胞的增殖。     

Stable transfer and expression of exogenous human globin genes in human erythroleukemia (K562) cells.

To study the expression of globin genes in human cells, human epsilon-globin genes were transferred into a K562 cell line, Bos, which synthesizes very low amounts of epsilon-globin mRNA. A plasmid (pSV2neo-epsilon) containing a complete epsilon-globin gene and 2 kilobases (kb) of 5' flanking DNA as well as a neomycin-resistance gene and a simian virus 40 origin of replication was transfected into Bos cells; the compound G418, a neomycin analogue, was used to select transformed cells. The presence of unique bands by DNA restriction analysis shows that 11 of 14 of the G418-resistant clones have at least one copy of an integrated epsilon-globin gene. RNA expression measured by RNA blotting shows significantly more epsilon-globin mRNA sequences than in untransfected Bos cells in 10 of 11 lines; in most lines, epsilon-globin mRNA was additionally increased in the presence of hemin. In two lines, epsilon-globin mRNA expression with hemin was comparable to that of a high epsilon-globin producing cell line, K562 clone 2. The one G418-resistant line without epsilon-globin genes had no epsilon-mRNA expression. The high epsilon-mRNA expression in several of the lines suggests that exogenous epsilon-globin genes with only 2-kb 5' flanking DNA may be sufficient to be appropriately expressed in these homologous erythroid cells. These results have implications for the potential success of transfer of normal human genes to human bone marrow cells as an approach to the treatment of inherited anemias.     

柴胡皂甙d诱导K562细胞凋亡的差异基因筛选

目的 筛选柴胡皂甙d(SSd)诱导人白血病K562细胞凋亡的差异基因,为白血病的治疗奠定基础.方法 用Agilent Human 1A寡核苷酸芯片检测SSd处理前后K562细胞的基因表达谱变化,并筛选SSd诱导K562细胞凋亡的相关基因.结果 359个基因发生显著性变化,其中表达上调基因138个,表达下调基因221个.结论 SSd诱导K562细胞凋亡是一个多基因参与的过程,本文筛选出的差异基因有望成为人白血病治疗的靶基因.     

Identification and quantitation of embryonic and three types of fetal hemoglobin produced on induction of the human pluripotent leukemia cell line K-562 with hemin

The hemoglobins synthesized by the pluripotent K-562 leukemia cell line of human origin after induction with hemin have been isolated by DEAE-cellulose chromatography and characterized by electrophoresis, high pressure liquid chromatography, and a radio-immunological assay. Six hemoglobin zones have been observed with the following likely compositions. Zone 1: α₂?₂, or Hb Gower-2; zone 2: ζ ₂?₂, or Hb Gower-1; zone 3: ζ₂γ₂, or Hb Portland-I; zone 4: Hb F, or α₂γ₂; zone 5: a mixture of acetylated Hb Portland-I and Hb F; zone 6: Hb Bart's, or γ₄. The embryonic Hbs (zones 1, 2, and 3) constituted 50%–75% of the total Hb present; the quantities varied from one experiment to the other. Both Hb Gower-1 and Hb Gower-2 were present. The γ chain was heterogeneous and contained the ^G_γ,^Aγ^I, and ^Aγ^T types in a ratio of about 4:2:1, indicating a heterozygosity for the Ile → Thr substitution at position γ75. The methodology used can be applied for additional studies evaluating quantitative changes in Hb types due to in vitro manipulations.     

Differentiation and multidrug resistance in response to drug treatment in the K562 human leukaemia cell line

Summary. The relationship between differentiation and P-glycoprotein expression in response to chemotherapeutic drugs was studied in the K562 human leukaemia cell line by treatment with low, but clinically achievable levels of vinblastine and epirubicin. Resistant sublines were easily generated with the multidrug resistant phenotype being expressed in response to drug treatment as low as 1 ng/ml vinblastine and 10 ng/ml epirubicin. These sublines showed stable but heterogeneous expression of P-glycoprotein as revealed by immunocytochemistry, and confirmed by cloning. This heterogeneity was maintained over 18 months with intermittent drug treatment. While selection for resistance induced erythroid and myeloid differentiation, expression of P-glycoprotein was not correlated with the stem cell antigen CD34 or with specific markers of erythroid or myeloid differentiation.     

Determination of IGF-II levels in human serum using the erythroleukemia cell line K562

A homologous radioreceptor assay (RRA) has been developed for Insulin-like Growth Factor II (IGF II) using the human erythroleukemia cell line K562. These cells have binding sites for insulin and IGF-II but not for Insulin-like Growth Factor I (IGF I). All samples were dissociated and separated from binding proteins by gel filtration at acidic pH. In healthy adults the mean serum level of radioreceptor assayable IGF II (RRA-IGF II) and 95% confidence limits were 965 ng/ml and 717-1299 ng/ml, respectively. The mean level in GH deficient patients was significantly lower (p less than 0.001) compared with healthy subjects whereas no change was found in patients with acromegaly and uremia. Slightly lowered levels of RRA-IGF II were found in one patient with a tumor induced hypoglycemia.