Selective cytotoxicity of 3-amino-l-tyrosine correlates with peroxidase activity

Summary In the presence of 3-amino-l-tyrosine (3-AT), abundant brown pigment forms in human HL-60 cells, but not in a variety of other cell lines, which are reported to be lower in mean myeloperoxidase (MPO) content than HL-60. Cells were assessed for peroxidase activity with an ABTS-based colorimetric assay and compared to values obtained with known amounts of human myeloperoxidase. HL-60 cells were estimated to contain the equivalent of 37.1 ng myeloperoxidase/10⁶ cells versus 26.1 and 5.0 ng/10⁶ cells for human K562 and murine RAW 264.7 cell lines, respectively. HL-60 cells exhibited a nearly 60% inhibition of proliferation and >70% reduction in cell viability after 4 d of culture in the presence of 100 μg 3-AT per ml. Higher concentrations of 3-AT (up to 400 μg/ml) for 4 d reduced HL-60 proliferation by 80% and decreased viability to 1–3%. Comparable levels of cytotoxicity were achieved in KG-1 cells after 7 d with 200 or 400 μg 3-AT per ml. K562 cells exhibited a 40% reduction in cell number after 7 d with 400 μg 3-AT per ml, but concentrations less than 400 μg/ml did not significantly affect K562 proliferation. K562 viability remained unchanged with doses of 3-AT up to 400 μg/ml. RAW 264.7 cells exhibited unchanged viability and proliferation in the presence of 3-AT at concentrations up to 400 μg 3-AT per ml. K562, KG-1, and RAW 264.7 cells exhibited no evidence of brown pigment formation in the presence of 3-AT and medium containing 10% fetal bovine serum. However, RAW 264.7 cells that were converted to protein-free medium and exposed to 3-AT exhibited intense brown pigment in some cell nuclei. A high percentage of HL-60 cells treated with 3-AT exhibited membrane blebbing, pyknosis, and nuclear fragmentation, which was not observed among other 3-AT-treated cell lines. A mechanism involving toxic intermediates of peroxidase-mediated “aminomelanin” formation is hypothesized.     

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.     

Recombinant human parvovirus B19 vectors: erythrocyte P antigen is necessary but not sufficient for successful transduction of human hematopoietic cells

The blood group P antigen, known to be abundantly expressed on erythroid cells, has been reported to be the cellular receptor for parvovirus B19. We have described the development of recombinant parvovirus B19 vectors with which high-efficiency, erythroid lineage-restricted transduction can be achieved (S. Ponnazhagan, K. A. Weigel, S. P. Raikwar, P. Mukherjee, M. C. Yoder, and A. Srivastava, J. Virol. 72:5224-5230, 1998). However, since a low-level transduction of nonerythroid cells could also be detected and since P antigen is expressed in nonerythroid cells, we reevaluated the role of P antigen in the viral binding and entry into cells. Cell surface expression analyses revealed that approximately 75% of primary human bone marrow mononuclear erythroid cells and approximately 31% of cells in the nonerythroid population were positive for P antigen. Two human erythroleukemia cell lines, HEL and K562, and a human promyelocytic leukemia cell line, HL-60, were also examined for P antigen expression and binding and entry of the vector. HEL and K562 cells showed intermediate levels, whereas HL-60 cells demonstrated high levels of expression of P antigen. However, the efficiency of vector binding to these cells did not correlate with P antigen expression. Moreover, despite P antigen positivity and efficient viral binding, HEL, K562, and HL-60 cells could not be transduced with the vector. Low levels of P antigen expression could also be detected in two primary cell types, human umbilical vein endothelial cells (HUVEC) and normal human lung fibroblasts (NHLF). In addition, vector binding occurred in both cell types and was inhibited by globoside, indicating the involvement of P antigen in virus binding to these cells. These primary cells could be efficiently transduced with the recombinant vector. These data suggest that (i) P antigen is expressed on a variety of cell types and is involved in binding of parvovirus B19 to human cells, (ii) the level of P antigen expression does not correlate with the efficiency of viral binding, (iii) P antigen is necessary but not sufficient for parvovirus B19 entry into cells, and (iv) parvovirus B19 vectors can be used to transduce HUVEC and NHLF. These studies further suggest the existence of a putative cellular coreceptor for efficient entry of parvovirus B19 into human cells.     

Stimulation of sialidase activity during cell differentiation of human promyelocytic leukemia cell line HL-60

Sialidase activity of human promyelocytic leukemia cell line HL-60 was assayed by a modification of the fluorometric method using 4MU-NANA as a substrate. The pH optimum was 4.1 and the apparent Km value was 0.10 mM. When the cells were induced to differentiate into granulocytes by either retinoic acid or DMSO, sialidase activity increased markedly. After incubation of HL-60 cells with 1 μM retinoic acid for 6 days and with 1.3% DMSO for 8 days, 91% and 75% of total cells, respectively, differentiated into morphologically mature myeloid cells and the sialidase activity increased to 2.5–2.7 times as much as that of the corresponding controls. In other human myeloid leukemia cell lines, K562 and KG-1, the sialidase activity was found to be 1.5- and 3.8-fold that of HL-60, respectively.     

2,7-Dihydro-3H-pyridazino[5,4,3-kl]acridin-3-one derivatives, novel type of cytotoxic agents active on multidrug-resistant cell lines. Synthesis and biological evaluation

We have earlier postulated that the presence of a pyridazone ring fused with an anthracenedione moiety resulted in the analog's ability to overcome multidrug resistance of tumor cells [J. Med. Chem.1999, 42, 3494]. High cytotoxic activity of obtained anthrapyridazones [Bioorg. Med. Chem.2003, 11, 561] toward the resistant cell lines, prompted us to synthesize the similarly modified acridine compounds. A series of pyridazinoacridin-3-one derivatives (2b-h) were prepared from the reaction of 9-oxo-9,10-dihydroacridine-1-carboxylate with POCl(3), followed by addition of the appropriate (alkylamino)alkylhydrazines. In vitro cytotoxic activity toward sensitive and resistant leukemia cell lines: L1210, K562, K562/DX, HL-60, HL-60/VINC, and HL-60/DX, with various type of multidrug resistance (MDR and MRP) was determined. The compounds studied exhibited in comparison to the reference cytostatics (DX, MIT) desirable very low resistance indexes (RI). Variations have been observed depending upon the substituent and the type of drug exporting pump. The cytotoxic activities of examined compounds, as well as of model anthrapyridazone derivative PDZ, were lower than those of reference drugs (DX, MIT) due to their diminished affinity to DNA.     

Cadmium- and chromium-induced oxidative stress, DNA damage, and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells

Sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] are both cytotoxic and mutagenic. This study examined the toxic and apoptotic potentials of these two cations on three cell types in vitro, namely, human chronic myelogenous leukemic (CML) K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells. The cells were incubated with 0-100 microM concentrations of the two cations for 0, 24, or 48 hours at 37 degrees C. Both Cr(VI) and Cd(II) induced changes in intracellular oxidized states of cells, which were detected using laser scanning confocal microscopy. Cell cycle modulation and apoptosis of the K562 cells by Cr(VI) and Cd(II) were determined by flow cytometry. Significant decreases in the G2/M phase were observed in the Cr(VI) and Cd(II) treated CML cells compared with untreated cells. At 12.5 microM, Cr(VI) induced greater apoptosis in K562 cells as compared with Cd(II). In the K562 cells, 2.2- and 3.0-fold increases in DNA fragmentation were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.2- and 1.7-fold increases in DNA fragmentation were observed with Cd(II). Furthermore, approximately 2.7- and 4.9-fold increases in cytochrome c reduction were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.6- and 3.3-fold increases in cytochrome c reduction were observed with Cd(II), demonstrating enhanced production of superoxide anion. Approximately 3.1 to 6-fold increases in hydroxyl radical production were observed following incubation of the K562 cells with these cations at 12.5 and 25 microM concentrations. These results in K562 cells were compared with promyelocytic leukemic HL-60 cells and normal human peripheral blood mononuclear cells. More pronounced effects were observed on K562 and HL-60 cells, and much lesser effects were observed on normal human peripheral blood mononuclear cells. The results demonstrate that both cations are toxic, producing oxidative tissue damage and apoptosis. Furthermore, more drastic effects were observed on K562 and HL-60 cells as compared with normal human peripheral blood mononuclear cells.     

Evidence of intracellular and trans-acting differentiation-inducing activity in human promyelocytic leukemia HL-60 cells: its possible involvement in process of cell differentiation from a commitment step to a phenotype-expression step

We previously reported that human promyelocytic leukemia HL-60 cells, when treated with various inducers in magnesium-deficient medium, became committed to differentiate but did not express the differentiation-related phenotypes (Okazaki et al., J. Cell. Physiol., 131:50-57, 1987). In the present study we demonstrated the existence of an intracellular differentiation-inducing activity (int-DIA) in differentiation-committed phenotype-nonexpressing HL-60 cells by using cybrid formation between untreated HL-60 cells and cytoplasts from HL-60 cells treated in magnesium-deficient medium with 100 nM 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). Cell extracts from similarly treated HL-60 cells also showed int-DIA, which when added (10 mg total protein/ml) to culture of untreated HL-60 cells, could increase the percentages of nitroblue tetrazolium (NBT)- and nonspecific esterase (NSE)-positive cells from 1% to 53%, and from 0 to 32%, respectively. They also induced differentiation of human monoblastic leukemia U-937 cells and of human myeloblastic leukemia KG-1 cells but not of erythroleukemia K-562 cells. These results suggested that the int-DIA had a common effect on differentiation induction in several human myeloid cell lines and may be involved in inducing cells to proceed from a commitment to a phenotype-expression step during human myeloid cell differentiation.     

Clustering of endocytic organelles in parental and drug-resistant myeloid leukaemia cell lines lacking centrosomally organised microtubule arrays

Spatial organisation and trafficking of endocytic organelles in mammalian cells is tightly regulated and dependent on cytoskeletal networks. The dynamics of endocytic pathways is modified in a number of diseases, including cancer, and notably in multidrug resistant (MDR) cells that are refractory to the effects of several anti-cancer agents. These cells often upregulate expression of drug-efflux pumps but this may be synergistic with alternative resistance mechanisms including increased acidification of endocytic organelles that enhances vesicular sequestration of weak-base anti-cancer drugs such as daunorubicin away from their nuclear target. Here, we characterised the distribution of sequestered daunorubicin in commonly used leukaemia cell lines, HL-60, K562, KG1a and the multidrug resistant HL-60/ADR line, and related this to the spatial distribution of their endocytic organelles and microtubule networks. HL-60 and KG1a cells contained microtubule arrays emanating from organising centres, and their endocytic organelles and daunorubicin labelled vesicles were scattered throughout the cytoplasm. HL-60/ADR and K562 cells showed extensive clustering of early and recycling endosomes, late endosomes, lysosomes and daunorubicin to a juxtanuclear region but these cells lacked microtubule arrays. Microtubular organisation within these clustered regions was however, required for spatial tethering of endocytic organelles and the Golgi, as treatment with nocodazole and paclitaxel had major effects on their distribution. HL-60 and HL-60/ADR cells had similar lysosomal pH of <5.0 and overall these findings suggests a general relationship between the absence of microtubule arrays and the propensity of leukaemia cell lines to cluster endocytic organelles and daunorubicin into the juxtanuclear region.     

DNA甲基化通过锌指蛋白185调控慢性粒细胞白血病细胞的增殖

锌指蛋白185(ZNF185)属于LIM结构域蛋白,参与细胞的增殖和分化,在多种肿瘤细胞中具有抑癌基因的功能.ZNF185在正常人血液系统细胞中高表达,但目前对白血病细胞的作用未见研究.采用Western blot检测人外周血中性粒细胞、急性粒细胞白血病细胞系HL-60和慢性粒细胞白血病细胞系K562细胞中ZNF185的表达,发现ZNF185在HL-60和K562细胞中的表达水平显著低于外周血中性粒细胞.为了阐明ZNF185对慢性粒细胞白血病细胞增殖的影响,从人外周血中性粒细胞克隆ZNF185编码序列,转染K562细胞,MTT检测细胞增殖,发现过表达ZNF185显著抑制K562细胞的增殖.甲基化特异PCR分析表明:ZNF185启动子在HL-60和K562细胞中高甲基化,用5-氮杂-2′-脱氧胞苷处理K562细胞,促进ZNF185的表达,显著抑制细胞增殖.研究结果表明,ZNF185启动子高甲基化导致其在K562细胞中的表达降低和细胞增殖抑制作用减弱.可能是慢性粒细胞白血病发生或发展的原因之一.     

Biologic Activity of a Nucleotide Conjugate Between Mitomycin C and Cytarabine Monophosphate

Abstract

A dual prodrug conjugate between the antimetabolite cytarabine monophosphate and the alkylating agent 2,7-diaminomitosene (derived from mitomycin C), cytaramycin, was synthesized and tested for antileukemic activity in sensitive and resistant tumors. The compound was active against parental L-1210, CCRF-CEM, HL-60 and K-562 leukemia cells but did not overcome resistance in sublines developed for (1) multidrug resistance (L-1210/MDR and K-562-R) or (2) for cytarabine resistance (CCRF-CEM/ARA-C and HL-60/ARA-C). Alkaline DNA elution tests demonstrate a predominance of strand breaking activity due to the cytarabine moiety, and a lesser degree of DNA crosslinking, due to the mitosene moiety. The conjugate was active in mice bearing P-388 leukemia (80% increased lifespan), but was not more effective than mitomycin C alone in mice bearing a cytarabine-resistant L-1210 cell line (38% to 31% increased lifespan). These findings suggest that mitomycin nucleotide conjugates do not overcome resistance to the parent antimetabolites.     

Biosynthesis of glycosphingolipids by human myeloid leukemia cells

We have performed comparative studies of the neutral glycosphingolipids synthesized by three human myeloid leukemia cell lines, K562, KG1, and HL-60, which were metabolically labeled with [14C]galactose, to evaluate changes in neutral glycosphingolipid synthesis with myeloid cell differentiation. Individual neutral glycosphingolipids containing one to four sugars were purified by a combination of the following methods: diethylaminoethyl-Sephadex column chromatography, acetylation-Florisil column chromatography, and high-performance liquid chromatography using an Iatrobead column. Compounds with one sugar were analyzed by thin-layer chromatography on borate plates. This analysis showed that HL-60 cells synthesize only glucosylceramide, whereas K562 and KG1 cells synthesize predominately glucosylceramide, but also a small amount of galactosylceramide. Compounds with two to four sugars were characterized by treatment with exo- and endoglycosidases. The results showed that K562 and KG1 cells are similar to cells from patients with acute leukemia in expressing two series (globo and neolacto) of natural glycosphingolipids, whereas the HL-60 cells are similar to mature human myeloid cells in expressing only one series (neolacto). Therefore, human myeloid leukemia cells blocked at different stages of differentiation vary in their ability to synthesize neutral glycosphingolipids.     

Optimization of a retroviral vector for transduction of human CD34 positive cells

Human stem and progenitor cells have recently become objects of intensive studies as an important target for gene therapy and regenerative medicine. Retroviral vectors are among the most effective tools for genetic modification of these cells. However, their transduction efficiency strongly depends on the choice of the ex vivo transduction system. The aim of this study was to elaborate a system for retroviral vector transduction of human CD34 positive cells isolated from cord blood. The retroviral vector pMINV EGFP was chosen for transduction of two human erythroblastoid cell lines: KG-1a (CD34 positive) and K562 (CD34 negative). For vector construction, three promoters and two retroviral vector packaging cell lines were used. To optimize the physicochemical conditions of the transduction process, different temperatures of supernatant harvesting, the influence of centrifugation and the presence of transduction enhancing agents were tested. The conditions elaborated with KG-1a cells were further applied for transduction of CD34 positive cells isolated from cord blood. The optimal efficiency of transduction of CD34 positive cells with pMINV EGFP retroviral vector (26% of EGFP positive cells), was obtained using infective vector with LTR retroviral promoter, produced by TE FLY GA MINV EGFP packaging cell line. The transduction was performed in the presence of serum, at 37 degrees C, with co-centrifugation of cells with viral supernatants and the use of transduction enhancing agents. This study confirmed that for gene transfer into CD34 positive cells, the detailed optimization of each element of the transduction process is of great importance.     

Different effects of an oligonucleotide uptake stimulating protein on leukemic cells in their primitive and differentiated state

The single stranded [³H]oligonucleotide uptake by HL-60 human promyelocyte and K562 human erythroleukemia cells was stimulated 20–45-fold by DUSF (DNA uptake stimulating protein), and this effect was drastically reduced (to 1.6–13×) if the cells were induced to differentiate. The oligonucleotide uptake stimulating effect of DUSF was not altered in HL-60 and K562 cells, if the proliferation of the cells was inhibited by hydroxyurea (HU) treatment. The oligonucleotide uptake by separated granulocytes and mononuclear cells from healthy donors was not stimulated by DUSF, while the uptake of oligonucleotides by myeloid and lymphoid leukemic cells was greatly stimulated (10–15×). The uptake of oligonucleotides by differentiated mononuclear cells of healthy donors could not be stimulated by DUSF, but the oligonucleotide uptake was greatly increased (11×) by DUSF if the cells were subjected to blast transformation.     

The ability to cleave 28S ribosomal RNA during apoptosis is a cell-type dependent trait unrelated to DNA fragmentation

Discrete cleavages within 28S rRNA divergent domains have previously been found to coincide with DNA fragmentation during apoptosis. Here we show that rRNA and DNA cleavages can occur independently in apoptotic cells, i.e. that the previously observed correlation is likely to be coincidental. In HL-60 cells, apoptosis with massive DNA fragmentation could be induced without any signs of rRNA cleavage. The opposite situation; rRNA cleavage without concomitant internucleosomal DNA fragmentation, was found in okadaic acid-treated Molt-4 cells. Other leukemia cell lines underwent apoptosis either without (K562 and Molt-3) or with (U937) both forms of polynucleotide cleavage. In K562 cells transfected with a temperature-sensitive p53 mutant, internucleosomal DNA fragmentation but not 28S rRNA cleavage was inducible by wild-type p53 expression. The absence of apoptotic rRNA cleavage in some cell types suggests that this phenomenon is tightly regulated and unrelated to DNA fragmentation or a presumed scheme for general macromolecular degradation in apoptotic cells.     

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.