Differentiation of Mouse and Human Myeloid Leukemia Cells Induced by an Antitumor Antibiotic,Ascofuranone

Mouse myeloid leukemia cells, M1, were induced to differentiate into phagocytes by treatment with ascofuranone (AF). AF also induced differentiation of human promyelocytic leukemia HL60 cells and human erythroid leukemia K562 cells into granulocytes and erythrocytes, as detected by nitroblue tetrazolium reducing activity and benzidine staining, respectively.

The antibiotic enhanced acetate incorporation of K562 cells. The increase was not observed with the cells of HL60 and two human B lymphoma lines, Daudi and Raji. The increase was diminished by the addition of a glycolysis inhibitor, deoxyglucose. Inhibitors of respiration, antimycin and sodium azide, also enhanced acetate incorporation of K562 cells specifically, which was diminished by the addition of deoxyglucose. Furthermore, antimycin induced differentiation of K562 and HL60 cells. These results suggest a possible relationship between cell differentiation and inhibition of respiration.     

Mannosylerythritol lipid induces granulocytic differentiation and inhibits the tyrosine phosphorylation of human myelogenous leukemia cell line K562

Mannosylerythritol lipid (MEL), which induced granulocytic differentiation of human promyelocytic leukemia cell line HL60, also induced differentiation of human myelogenous leukemia cell line K562. MEL inhibited insulin-dependent cell proliferation and induced leukocyte esterase activity of K562 cells. MEL markedly increased the differentiation-associated characteristics in granulocytes, such as nitroblue tetrazolium (NBT) reducing ability, expression of Fc receptors, and phagocytic activity of K562 cells. The tyrosine phosphorylation in K562 cells inhibited by MEL. These results suggest that MEL directly down-regulates the tyrosine kinase activities in K562 cells to inhibit the cell proliferation and to induce the differentiation. This revised version was published online in June 2006 with corrections to the Cover Date.     

K562 leukemia cells transfected with the human c-fes gene acquire the ability to undergo myeloid differentiation

Expression of the proto-oncogene p93c-fes and its associated tyrosine kinase activity is marked in mature granulocytes, monocytes, differentiated HL-60 leukemia cells, and leukemia cell lines KG-1, THP-1, HEL, and U-937, which can be induced to differentiate along the granulocyte/monocyte pathway. Conversely, p93-c-fes expression is absent in the K562 cell line, which is resistant to myeloid differentiation. Upon transfection and clonal selection of K562 cells using a mammalian expression vector containing the 13-kilobase pair c-fes gene, c-fes mRNA was transcribed and p93-c-fes tyrosine activity kinase was expressed. Clones expressing c-fes underwent myeloid differentiation as assessed by the appearance of phagocytic activity, Fc receptors, nitro blue tetrazolium reduction, Mac-1 immunofluorescence, and lysozyme production. These results indicate that the expression of the c-fes protooncogene and its associated tyrosine kinase activity plays a major role in the initiation of myeloid differentiation.     

Interaction of the photosensitizer 13,15-<Emphasis Type="Italic">N</Emphasis>-(3′-hydroxypropyl)cycloimide chlorin p<Subscript>6</Subscript> with normal and cancerous blood cells

The interaction of 13,15-N-(3′-hydroxypropyl)cycloimide chlorin p₆ (CIC) with normal blood cells and human myeloid leukemia K562 and HL60 cells was studied. CIC was found to be bound by the erythrocyte membrane but did not penetrate into the cytoplasm. It is characterized by a diffuse distribution in the cytoplasm of normal leukocytes, whereas its diffuse distribution in K562 and HL60 cells is accompanied by perinuclear accumulation and binding to the plasma membrane. The average cytoplasmic concentration corresponding to the CIC accumulation in leukemic cells at saturation is 2.2 to 2.6 times higher than that in normal leukocytes. CIC is more intensely accumulated in granulocytes than in lymphocytes. The kinetics of the cellular uptake and efflux was characterized. The normal leukocytes and erythrocytes were found to be 1.5 times and 3 to 4 times less sensitive, respectively, to the photodynamic action of CIC than the K562 and HL60 cells.     

Lineage-related sensitivity to apoptosis in human tumor cells undergoing hyperthermia

In this study the role of hyperthermia as an apoptotic trigger was analyzed in four human tumor cell lines: HL60, U937, DOHH₂, and K562. These cell lines were chosen because of their well known and different expression of bcl-2 and bcr-abl genes, the expression of which is known to be an antiapoptotic condition. HL60 and U937 cells were strongly susceptible to heat exposure, while DOHH₂ cells were weakly sensitive and K562 cells were resistant, thus suggesting a possible gene involvement in this type of programmed cell death. The mechanisms underlying this apoptosis were investigated by flow cytometry, agarose gel electrophoresis, and light and electron microscopy. A subdiploid peak and DNA laddering, both of which are parameters specifically correlated to programmed cell death, were present in HL60 and U937 and, even if less evident, in DOHH₂ cells undergoing hyperthermic treatment, and were absent in K562 cells. In addition, DNA single-strand cleavage was revealed by in situ nick translation, observed by confocal microscopy. Morphological analysis confirmed these results and revealed the typical chromatin changes, followed by the appearance of micronuclei and apoptotic bodies. Accepted: 26 November 1999     

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     

Prediction of VEGF mRNA antisense oligodeoxynucleotides by RNA structure software and their effects on HL60 and K562 cells

Seven antisense oligodeoxynucleotides were selected using RNA structure 3.7 software and the principle of low overall DeltaG (free energy). Their effects on cell growth, VEGF protein expression and apoptosis in HL60 and K562 leukemic cells were examined, and cell numbers and viability were assessed using trypan blue dye exclusion, MTT, ELISA and flow cytometry. The results showed that six of the seven antisense sequences inhibited the cell growth and down-regulated VEGF protein expression significantly. Endogenous VEGF plays an important role in the proliferation of HL60 and K562 leukemic cells. RNA structure software provides a rapid and efficient way of identifying effective antisense oligodeoxynucleotides.     

Correlation between the oscillatory and adhesion activities in erythroid cells

The attachment kinetics of erythroid cells, such as human erythrocytes, their saponin ghosts, and erythroleukemic cells K562 to a glass surface has been studied in the presence of substances inhibiting spontaneous fluctuations of cell membranes. It has been shown that wheat germ agglutinin (WGA) slows down the attachment kinetics of K562 cells, as is the case in intact erythrocytes. Concanavalin A (Con A), which inhibits the attachment of erythrocytes to glass does not affect the adhesion of K562 cells to glass due to the absence of band 3 proteins in the membranes of K562 cells. Both lectins slow down the adhesion rate of saponin ghosts of human erythrocytes, as it takes place in intact erythrocytes. Suramin and the anionic dye ANS bind specifically to the actin protofilaments of the erythrocyte skeleton and also inhibit cell adhesion to glass. At the same time, these substances do not affect the oscillatory and adhesion activities of intact erythrocytes due to the impermeability of erythrocyte membranes for these drugs. The results obtained allow the conclusion that inhibition of erythrocyte adhesion by lectins is due to lectin binding to different constituents of the erythrocyte membrane--sialic acid moieties of glycophorin in the case of WGA and band 3 proteins in the case of Con A. The most probable mechanism of erythrocyte and K562 cell attachment to glass is the formation of the so-called local contacts between cells and the glass surface. It is also suggested that the cell surface oscillations facilitate the formation of cell contacts.     

7-Hydroxystaurosporine (UCN-01) Induces Apoptosis in Human Colon Carcinoma and Leukemia Cells Independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.     

Tetrahydrocannabinol inhibits adenyl cyclase in human leukemia cells

Delta 9-tetrahydrocannabinol has been shown to induce incomplete maturation in ML2 human leukemia cell lines. We extend the observation of its induction of morphologic maturation to HL60 cells and of its induction of growth restriction to HL60 and K562 cells. We show that tetrahydrocannabinol reduces the cyclic AMP content of ML2 cells. Finally we demonstrate that this agent inhibits adenyl cyclase activity in ML2 cell membrane-enriched fractions. This finding in myeloid cells is compatible with one hypothesis of cannabinoid action in neuronal cells.     

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.     

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.     

Bufalin as a potent inducer of differentiation of human myeloid leukemia cells.

Bufalin was found to be a potent inducer of differentiation in human erythroleukemia K562 cells by examination of various differentiation markers (as assessed by the morphology, histochemistry, and the abilities to phagocytose latex particles, to reduce nitro-blue tetrazolium and to develop Fc receptors). Bufalin, at a concentration as low as 10 nM, also produced a strong differentiation-inducing activity in three other human leukemia-derived cell lines (human promyelocytic HL60, monoblastic U937 and myeloblastic ML1). Treatment of K562 cells with other cardiotonic steroids, such as cinobufagin, ouabain and digitoxigenin, at the concentration of 10 nM for four days resulted in weak or no effect on the cells. These findings suggest that bufalin might have potentiality as a new agent in the differentiation therapy for human myelogenous leukemia.     

Studies on the mechanism of specificity of human natural killer cells for tumor cells: correlation between target cell transferrin receptor expression and competitive activity

Previous studies to determine the nature of the specificity of natural killer (NK) cells for leukemic cells indicated that functional transferrin (Tf) receptors may be one of the determinants recognized by NK cells. To further investigate these observations, the relationship between cellular Tf receptor expression and ability to compete with a control K562 cell preparation in a standard chromium release assay was studied. K562 cells were selected at different phases of growth by removing cells from tissue culture at 1, 3, and 5 days postfeeding. Under these conditions, K562 cells, respectively, displayed relatively high, medium, and low numbers of Tf receptors and corresponding competitive activity against a control K562 cell preparation. K562 cells were modified by either trypsin, heat, or sodium butyrate (differentiation inducer) pretreatment. An NK-resistant clone was also studied. There was a good correlation between Tf receptor expression and cold competitive activity of the above K562 cell preparations (r = 0.82, P less than 0.01). The different tumor target cell lines, K562, Molt-4, Raji, HL-60, and MeWo, which would be expected to express different ranges of specificity, did not show a significant correlation between Tf receptor expression and their cold competitive activities against Cr-51-labeled K562 cells. Rabbit reticulocytes which express high numbers of Tf receptors were tested for their ability to compete with K562 cells for NK cells. These cells were able to compete with K562 cells while mature rabbit red blood cells which do not express Tf receptors did not compete well. These findings support the contention that the Tf receptor may be involved in NK cell recognition of some tumor cells.     

Temporal relationships between induced changes in c-myc mRNA abundance, proliferation, and differentiation in HL60 cells

Changes in the relative abundances of c-myc mRNA have been related to changes in other parameters of differentiation (histochemical, clonogenic) during the course of the differentiation of HL60 cells to monocytes/macrophages or to granulocytes. Induction of differentiation to monocytes/macrophages was marked by a rapid rate of appearance of committed cells (80 to 90% in 24 hours) and a concomitant rapid loss of c-myc mRNA. Induction of granulocytic differentiation resulted in a much slower rate of appearance of committed cells (50% in 48 hours), and a much faster rate of loss of c-myc mRNA (tenfold in 1 hour). These data are consistent with there being a direct link between down-regulation of the expression of c-myc and the onset of proliferation arrest and monocytic differentiation, but show there is no such association of c-myc mRNA abundance and proliferation or differentiation during the maturation of HL60 granulocytes.