Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of leukemic cells to arsenic trioxide

Cell culture, tissue chemistry and flow cytometry were used to determine whether antisense bcl-2 oligodeoxynucleotides enhanced the sensitivity of leukemia cells to arsenic trioxide. A combination of arsenic trioxide with antisense bcl-2 oligodeoxynucleotides inhibited cell growth, induced apoptosis and induced bcl-2 protein expression in K562 and NB4 leukemic cells more significantly than either arsenic trioxide or the oligodeoxynucleotides on their own (P<0.01). Thus, bcl-2 antisense oligodeoxynucleotides increase the sensitivity of leukemic cells to arsenic trioxide. Combined use of the two agents could be a novel and attractive strategy in leukemia treatment.     

Electrophoretic pattern of NADPH-dependent oxidoreductive activities in K 562 and HL 60 leukemic cell lines

Morphological and functional differentiation of hemopoietic cells is accompanied by the expression of lineage-specific protein markers. NADPH-oxidoreductive enzymatic activities in HL 60 and K 562 leukemic cell lines, compared with granulocytes and erythrocytes, show a NADPH-oxidizing and a NADPH-diaphorase activity. The oxidizing activity, absent in erythrocytes, has the same electrophoretic migration in HL 60 cells and granulocytes while it is different in K 562 cells. The diaphorase, absent in HL 60 cells and granulocytes, has the same migration in erythrocytes and K 562 cells, although with a slightly different quantitative expression. K 562 cells induced to differentiation with arabinofuranosylcytosine show the appearance of a band of NADPH-oxidizing activity of granulocytic type, together with the major band found in these cells.     

Arachidonate 5-lipoxygenase inhibitors show potent antiproliferative effects on human leukemia cell lines

Cirsiliol and AA861, specific arachidonate 5-lipoxygenase inhibitors, showed potent antiproliferative effects on human leukemic cell lines K562, Molt4B and HL60. On the other hand, HeLa cells were not affected by these drugs. In the inhibitor treated and growth retarded leukemia cells, the rates of synthesis of DNA, RNA and protein were markedly decreased. These results suggested that arachidonate 5-lipoxygenase or leukotrienes would play essential roles in cellular functions of leukemic cells.     

Bcr3/abl2和VEGF基因反义寡核苷酸联用增强对K562细胞的生长抑制作用

目的:研究BCRABL和VEGF反义寡核苷酸联用对K562细胞株的作用及其相互作用的影响。方法:设计针对bcr3/abl2和VEGF的反义寡核苷酸(ASODNs),应用脂质体Oligofectamine作为转染载体。在转染后72h进行台盼蓝染色细胞计数;建立裸鼠K562移植瘤动物模型,瘤内注射ASODNs,观察肿瘤体积生长变化,组织学检测肿瘤血管密度和肿瘤细胞凋亡情况。结果:转染后72h,各实验组与空白组相比,细胞增殖抑制率分别为13.47%(ASOB3/A2组),12.79%(ASOVEGF组)和41.55%(半量联合治疗组)。经过4次治疗后,与对照组相比,肿瘤生长抑制率分别为23.18%(ASOB3/A2组),17.28%(ASOVEGF组)和57.83%(半量联合治疗组)。联合治疗组肿瘤生长速率显著低于单一治疗组,伴随明显的肿瘤细胞凋亡增加和肿瘤血管密度减少。结论:双基因反义寡核苷酸联合应用协同抑制K562细胞增殖,抗肿瘤作用明显优于单一治疗组,可为CML基因治疗提供一项新策略。     

Bcr-abl translocation can occur during the induction of multidrug resistance and confers apoptosis resistance on myeloid leukemic cell lines

Apoptosis was studied in parental and mdr-1 expressing U937, HL60 and K562 myeloid leukemic cell lines using mdr unrelated inducers of apoptosis such as Ara-C, cycloheximide, serum deprivation, ceramide, monensin and UV irradiation. Apoptosis was efficiently induced by all these treatments in U937 and HL60 cells while K562 cells exhibited an apoptosis-resistant phenotype except with UV and monensin. The pattern of apoptosis resistance in mdr-1 expressing U937 (U937-DR) and HL60 (HL60-DR100) was similar to that presented by K562. This apoptosis-resistant phenotype of mdr cells was not overcome by concentrations of verapamil inhibiting the P-gp 170 pump. The acquisition of this phenotype was posterior to the mdr-1 expressing phenotype since a HL60-DR5 variant, selected at the beginning of the induction of resistance, presented a low level of mdr-1 expression without resistance to apoptosis. The variations observed in the Fas (CD95) expression between sensitive and resistant cells were not sufficient to account for apoptosis resistance. However, a high expression in Abl antigen was found in all the apoptosis-resistant cells. RT-PCR and Western blot analysis showed that this increase in Abl antigen content was accompanied by the expression in U937-DR and HL60-DR100 cells of a hybrid bcr/abl mRNA and a 210 kD Bcr/Abl protein which was constitutive in K562. This expression was due to the translocation of abl and the amplification of the bcr-abl translocated gene. These results are in agreement with the role of Bcr/Abl tyrosine protein kinase as an inhibitor of apoptosis independently of the mdr-1 expression. They also suggest that translocation of the abl gene in the bcr region is a highly probable rearrangement in the mdr-1 expressing myeloid cells and that Bcr/Abl tyrosine kinase effect on apoptosis needs the regulation of intracellular pH and is inactive against UV-induced apoptosis.     

Downregulation of Bcr-Abl in K562 cells restores susceptibility to apoptosis: characterization of the apoptotic death

We examined the susceptibility of a variety of human leukemic cell lines to the induction of apoptosis. K562, a chronic myelogenous leukemic cell line which expresses the bcr-abl fusion gene, was found to be extremely resistant to apoptosis, irrespective of the inducing agent. This resistance can be attributed to the deregulated Abl kinase activity of bcr-abl, as downregulation of its expression using antisense oligodeoxynucleotides targeted to the beginning of the abl sequence in this chimeric gene rendered these cells susceptible to cytotoxic drug-induced apoptosis. Examination of the morphological and biochemical features of apoptosis in K562 cells revealed the typical membrane blebbing and chromatin condensation associated with this form of cell death. In situ TdT-mediated end labeling of the DNA revealed the presence of strand breaks in the treated cells and field inversion gel electrophoresis revealed the presence of large 10-50 kb fragments. However there was an absence of oligonucleosomal DNA fragmentation, whether or not Bcr-Abl was expressed. Thus, while inhibition of expression of Bcr-Abl renders K562 cells susceptible to apoptosis, the absence of oligonucleosomal DNA fragmentation in these cells is independent of the function of this molecule.     

Synthesis of Bcl-2 in response to anthracycline treatment may contribute to an apoptosis-resistant phenotype in leukemic cell lines.

BACKGROUND: Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment. METHODS: U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot. RESULTS: High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype. CONCLUSIONS: These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.     

Accessory function and interleukin 1 production by human leukemic cell lines

Highly purified T lymphocytes do not proliferate in response to mitogens, unless adherent HLA-DR-positive monocytes are added to the culture. This accessory function (AF) of monocytes requires the release of interleukin 1 (IL 1). Cells from three human leukemic cell lines, K562, HL60, and U937, could very efficiently replace monocytes in a 72-hr mitogen-induced T cell proliferation assay. The AF was clearly related to precise maturational stages of these cells; the hematopoietic precursor K562 cells spontaneously exerted high AF, but lost this property when treated with differentiation inducers. On the contrary, the promyelocytic HL60 cells and the "histiocytic" U937 cells exhibited no spontaneous AF, but acquired this property when induced to differentiate along the granulocytic and/or monocytic pathway. Three leukemic cells could not only stimulate T cells to proliferate and produce IL 2 in the presence of mitogens, but also under appropriate culture conditions these cells could produce IL 1, which could not be distinguished from normal human monocyte derived IL 1 by gel filtration and isoelectric focusing. Moreover, analysis of phenotypic markers revealed that AF and production of IL 1 could be demonstrated in different cell types and therefore are not restricted to the monocytic lineage. No HLA-DR antigen could be detected on K562 and HL60 cells. Thus, the expression of the DR antigens is not required for AF and IL 1 production in response to mitogens. These three human leukemic cell lines will provide convenient sources of human IL 1.     

Antisense EDRF1 gene inhibited GATA-1 transcription factor DNA-binding activity in K562 cells

Our previous studies showed that EDRF1 influenced expression of a-globin mRNA and synthesis of hemoglobin in K562 cells and modulated self-renewal of K562 cells. To illuminate the function of EDRF1 in K562 cells, sense and antisense EDRF1 constructs were prepared and transfected into K562 cells. By using microarray and dot blot assay, 60 cytokine receptors and some oncogenes sharing important functions in cell proliferation and differentiation were investigated. The results of this study demonstrated that IL-6 receptor, GM-CSF receptor, c-Jun/c-Fos, c-Myc and c-kit genes were regulated by antisense EDRF1 expression. The regulation was confirmed by RNA blot assay. GATA-1 mRNA expression was modulated by EDRF1 gene transfection. Electrophoretic mobility shift assay suggested that the DNA-binding activity of GATA-1 was remarkably inhibited in K562 cells expressing EDRF1 antisense gene. DNA binding activity of NF-E2 was at the same level as control experiment. Therefore EDRF1 may play a role in erythroid pro     

α-Tomatine-Mediated Anti-Cancer Activity In Vitro and In Vivo through Cell Cycle- and Caspase-Independent Pathways

α-Tomatine, a tomato glycoalkaloid, has been reported to possess antibiotic properties against human pathogens. However, the mechanism of its action against leukemia remains unclear. In this study, the therapeutic potential of α-tomatine against leukemic cells was evaluated in vitro and in vivo. Cell viability experiments showed that α-tomatine had significant cytotoxic effects on the human leukemia cancer cell lines HL60 and K562, and the cells were found to be in the Annexin V-positive/propidium iodide-negative phase of cell death. In addition, α-tomatine induced both HL60 and K562 cell apoptosis in a cell cycle- and caspase-independent manner. α-Tomatine exposure led to a loss of the mitochrondrial membrane potential, and this finding was consistent with that observed on activation of the Bak and Mcl-1 short form (Mcl-1s) proteins. Exposure to α-tomatine also triggered the release of the apoptosis-inducing factor (AIF) from the mitochondria into the nucleus and down-regulated survivin expression. Furthermore, α-tomatine significantly inhibited HL60 xenograft tumor growth without causing loss of body weight in severe combined immunodeficiency (SCID) mice. Immunohistochemical test showed that the reduced tumor growth in the α-tomatine-treated mice was a result of increased apoptosis, which was associated with increased translocation of AIF in the nucleus and decreased survivin expression ex vivo. These results suggest that α-tomatine may be a candidate for leukemia treatment.     

Bcl-2 down-regulation causes autophagy in a caspase-independent manner in human leukemic HL60 cells

To understand the roles of bcl-2 for the survival of leukemic cells, we constructed human leukemic HL60 transformant lines in which full length bcl-2 antisense message was conditionally expressed by a tetracycline-regulatable expression system. Cell growth was completely inhibited after antisense message induction and massive cell death was induced. Electron microscopic examinations show that cells died by autophagy, but not by apoptosis. The morphology and the function of mitochondria remained intact: neither the reduction in mitochondrial membrane potential nor the nuclear translocation of AIF, a mitochondrial protein that translocates to nuclei in cases of apoptosis, was observed. Caspase inhibitors did not rescue bcl-2-antisense-mediated autophagy. Thus, bcl-2 is essential for leukemic cell survival and its down-regulation results in autophagy. Cell Death and Differentiation (2000) 7, 1263 - 1269.     

Antisense EDRF1 gene inhibited GATA-1 transcription factor DNA-binding activity in K562 cells

Our previous studies showed that EDRF1 influenced expression of α-globin mRNA and synthesis of hemoglobin in K562 cells and modulated self-renewal of K562 cells. To illuminate the function of EDRF1 in K562 cells, sense and antisense EDRF1 constructs were prepared and transfected into K562 cells. By using microarray and dot blot assay, 60 cytokine receptors and some oncogenes sharing important functions in cell proliferation and differentiation were investigated. The results of this study demonstrated that IL-6 receptor, GM-CSF receptor, c-Jun/c-Fos, c-Myc and c-kit genes were regulated by antisense EDRF1 expression. The regulation was confirmed by RNA blot assay. GATA-1 mRNA expression was modulated by EDRF1 gene transfection. Electrophoretic mobility shift assay suggested that the DNA-binding activity of GATA-1 was remarkably inhibited in K562 cells expressing EDRF1 antisense gene. DNA binding activity of NF-E2 was at the same level as control experiment. Therefore EDRF1 may play a role in erythroid proliferation and differentiation by affecting the interaction between GATA-1 and its cis-elements.     

Demonstration of the cell cycle positions of taxol-induced "asters" and "bundles" by sequential measurements of tubulin immunofluorescence, DNA content, and autoradiographic labeling of taxol-sensitive and -resistant cells

We used reliable and relatively inexpensive equipment to make sequential sets of measurements of antitubulin immunofluorescence, Feulgen staining, and autoradiography on the same cells. This was done to evaluate tubulin conformations, DNA content, and [3H]-thymidine incorporation in cell lines sensitive (HL60) and resistant (K562) to the novel anti-tubulin chemotherapeutic agent taxol. Numbers of cells with microtubule bundles have been found to correlate with sensitivity to taxol by clonogenic assay for several leukemic cell lines. We have found that cells with "asters" produced by taxol exposure are in mitosis and that cells with taxol-induced "bundles" are in G0/G1, S, and G2 phases. We further found that S-phase cells with microtubule bundles in both sensitive (HL60) and resistant (K562) cell lines were able to incorporate [3H]-thymidine after 4-hr exposure to taxol. As microtubule bundles and asters occur in cells of the same cell cycle phases in both lines, we conclude that the greater frequency of cells with microtubule bundles reported for sensitive cells after taxol treatment cannot result from drug exclusion nor from different effects of the drug on cell microtubules in these two leukemic lines.     

Immunological quantitation of phospholipid/Ca2+-dependent protein kinase and its fragments. Tissue levels, subcellular distribution, and ontogenetic changes in brain and heart

Levels of phospholipid/Ca2+-dependent protein kinase (protein kinase C, 80 kDa) and its presumed proteolytic fragments were quantified in a variety of animal tissues and cultured human leukemic cell lines (HL60 and K562) using an immunoblot analysis technique. Of many tissues examined, the rat brain and HL60 cells were by far the richest sources of the 80-kDa native enzyme, with its concentration estimated to be about 2-3 microM in both tissues. The major enzyme species detected in most tissues, however, was the 67-kDa fragment; the 80-kDa native enzyme was present in a smaller amount except in spleen which contained nearly equal amounts of both enzyme species. It was also found that HL60 and K562 leukemic cells contained the 50-kDa species instead of the 67-kDa species. A study of the subcellular distribution of the 80- and 67-kDa species showed the enzyme to be localized predominantly in the soluble fraction for some tissues (e.g. heart) and nearly equally distributed between soluble and particulate fractions in others (e.g. spleen). In the brain, however, the majority of the enzyme was present in the particulate fraction, in agreement with the findings made with immunocytochemical localization of the enzyme. The total enzyme content in developing rat brain and heart increased during the first 2 to 4 weeks following birth and decreased to 60% of peak levels in the adult. The present immunological method, showing for the first time that the tissue levels of phospholipid/Ca2+-dependent protein kinase and its fragments can be quantitated, would be useful for studies on the regulation of cellular events and pathophysiology of tissues thought to be involved in this multi-functional protein phosphorylation system.     

Cloning and Expression of the Functional Human Anti-vascular Endothelial Growth Factor (VEGF) Using the pcDNA3.1 Vector and the Human Chronic Myelogenous Leukemia Cell Line K562

In this study, the light chain (κ) and heavy chain (γ) sequences of the monoclonal antibody against vascular endothelial growth factor (VEGF) were sub-cloned into the eukaryotic pcDNA3.1 (+) (Hygro) and the pcDNA3.1 (+) (Neo) expression vectors using the traditional and homologous recombination methods. To express the antibody, the recombinant plasmids were transfected into the Chinese hamster ovary (CHO) and the K562 cell lines. The recombinant antibody was then purified using the protein A affinity chromatography. Furthermore, in order to demonstrate the inhibition of VEGF-induced mitogenesis of the recombinant antibody, the bovine aorta endothelial like cells were employed. The results showed specialization and conjunction of the recombinant antibody to the VEGF. It was also indicated that the antibody expression in the K562 cell lines was higher than the CHO cell lines. Furthermore, the in vitro VEGF inhabitation of the recombinant antibodies which were produced from the K562 cell line, and the CHO cell line, were similar. This proved that the K562 cell line is a good substitute for the CHO cell line in the production of the recombinant antibodies.     

TCP1 increases drug resistance in acute myeloid leukemia by suppressing autophagy via activating AKT/mTOR signaling

T-complex protein 1 (TCP1) is one of the subunits of chaperonin-containing T complex (CCT), which is involved in protein folding, cell proliferation, apoptosis, cell cycle regulation, and drug resistance. Investigations have demonstrated that TCP1 is a factor being responsible for drug resistance in breast and ovarian cancer. However, the TCP1 role in acute myeloid leukemia (AML) remains elusive. In the present study, we discovered that the TCP1 expression was elevated in AML patients and high TCP1 expression was associated with low complete response rate along with poor overall survival. TCP1 showed higher expression in the adriamycin-resistant leukemia cell line HL60/A and K562/A, comparing to their respective parent cells HL60 and K562 cells. TCP1 inhibition suppressed drug resistance in HL60/A and K562/A cells, whereas TCP1 overexpression in HL60 cells incremented drug resistance, both in vitro and in vivo. Mechanistic investigations revealed that TCP1 inhibited autophagy and adriamycin-induced cell apoptosis, and TCP1-mediated autophagy inhibition conferred resistance to adriamycin-induced cell apoptosis. Furthermore, TCP1 interacted with AKT and mTOR to activate AKT/mTOR signaling, which negatively regulates apoptosis and autophagy. Pharmacological inhibition of AKT/mTOR signal particularly activated autophagy and resensitized TCP1-overexpressing HL60 cells to adriamycin. These findings identify a novel role of TCP1 regarding drug resistance in AML, which advise a new strategy for overcoming drug resistance in AML through targeting TCP1/AKT/mTOR signaling pathway.Subject terms: Prognostic markers, Acute myeloid leukaemia