Human Myelogenous Leukemia K562 Cells Research Articles

Toxicity of Bread Wheat Lines Lacking Prolamins Encoded by theGli-B1/Gli-B5/Glu-B3andGli-D1/Glu-D3Loci in Coeliac Disease as Determined by their Agglutinating Activity

Peptic-tryptic (PT) digests of alcohol-soluble proteins from the flour of three mutant lines of bread wheat, lacking γ-gliadins, γ-gliadins and low molecular-weight glutenin subunits encoded by the Gli-B1/Gli-B5/Glu-B3 loci (line S. Pastore 4A), the Gli-D1/Glu-D3 loci (line Alpe 1I-) or both groups of loci (line DM 22166), were compared with those of the normal cultivars S. Pastore and Alpe 1 I for their agglutinating activities on human myelogenous leukemia K562(S) cells, agglutination being strongly associated with toxicity for the coeliac intestine. All of the genotypes tested contained A-type α-gliadins, which constituted about 19% of the gliadins in the S. Pastore and Alpe 1I cultivars, 24.5% in the S. Pastore 4A and Alpe 1I null lines, and 34.8% in the double mutant line, DM 22166, as determined by densitometric scanning of their acid polyacrylamide gel electrophoresis patterns. The minimal concentrations of PT digest required to agglutinate 100% of K562(S) cells were 73mg/l and 96mg/l, in the S. Pastore and Alpe 1I cultivars, respectively, compared with 146mg/l, 138mg/l and 200mg/l in the “null” lines, S. Pastore 4A, Alpe 1 I-and DM 22166, respectively. The results indicated that proteins other than α-gliadins are involved in the gluten-sensitive enteropathy.

Rapid Ca 2+ Mobilization in Single LGL Cells upon Interaction with K562 Target Cells--Role of the CD18 and CD16 Molecules

Changes in the intracellular Ca 2+ levels of human large granular lymphocytes (LGL), loaded with the fluorescent Ca 2+ indicator fura-2, have been studied upon addition of human chronic myelogenous leukemia K562 cells. The measurements, analyzed at the single-cell level using image analysis, indicate a rapid Ca 2+ mobilization in the effector cell upon interaction with its target cell. This mobilization appeared to be localized to an area within the effector cell that was in physical contact with target cells. The LGL responded with different kinetics in a transient manner and about 19% of them could undergo two or more responses. Data obtained from experiments performed with anti-CD16- and anti-CD18-pretreated LGL in the presence of target cells indicate that the CD16 and CD18 molecules are not likely to be the triggers of the Ca 2+ response, although they might participate in the recognition of the target cell.

Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA.

Lactoferrin, an iron-binding glycoprotein found in high concentrations in human milk and other epithelial secretions and in the secondary (specific) granules of neutrophils, is thought to be responsible for primary defence against microbial infection, mainly as a result of lactoferrin sequestration of iron required for microbial growth. Many other functions have been attributed to lactoferrin, including immunomodulation and cell growth regulation (reviewed in ref. 4). Some of these functions appear to be at least in part independent of the iron-binding activity of lactoferrin. It also has been consistently observed that lactoferrin interacts avidly with nucleic acids. Lactoferrin enhancement of the activity of natural killer and lymphokine-activated killer cells in vitro is inhibited by RNA and DNA. Lactoferrin taken up by K562 human myelogenous leukaemia cells appears in the nucleus where it is bound to DNA. We report here that binding of lactoferrin to DNA occurs under stringent conditions with distinct sequence specificity, and that interaction between lactoferrin and these sequences intracellularly leads to transcriptional activation.

Natural killer cell response in the inner ear.

Heterologous tumor cells (human chronic myelogenous leukemia K-562 cells) were injected into the perilymph and skin in guinea pigs in order to study the induction of NK cell activity in the inner ear. An in vitro transmission electron microscopic and immunohistochemical study showed that K-562 cells were attacked by guinea pig large granular lymphocytes. K-562 cells injected through the round window membrane were found to be targeted by NK cells emerging from surrounding venules after 7 to 9 days. During this time morhological changes occurred in the organ of Corti and stria vacularis. These findings suggest that the inner ear response to foreign cells induces activation and invasion of NK cells which occur relatively late compared with those in other organs such as skin.

Comparison of biochemical parameters of benzamide riboside, a new inhibitor of IMP dehydrogenase, with tiazofurin and selenazofurin

The biochemical and cytotoxic activities of the IMP dehydrogenase (IMPDH) inhibitors benzamide riboside, tiazofurin, and selenazofurin were compared. These three C-nucleosides exert their cytotoxicity by forming an analogue of NAD, wherein nicotinamide is replaced by the C-nucleoside base. The antiproliferative activities of these three agents were compared in a panel of 60 human cancer cell lines. To examine the relationship of benzamide riboside and selenazofurin to tiazofurin, COMPARE computer analysis was performed, and correlation coefficients of 0.761 and 0.815 were obtained for benzamide riboside and selenazofurin, respectively. The biochemical activities of these agents were examined in human myelogenous leukemia K562 cells. Incubation of K562 cells for 4 hr with 10 μM each of benzamide riboside, selenazofurin and tiazofurin resulted in a 49, 71, and 26% decrease in IMPDH activity with a concurrent increase in intracellular IMP pools. As a consequence of IMPDH inhibition, GTP and dGTP concentrations were curtailed. These studies demonstrated that selenazofurin was the most potent of the three agents. To compare the cellular synthesis of NAD analogues of these agents, K562 cells were incubated with 10 μM each of benzamide riboside, tiazofurin and selenazofurin after prelabeling the cells with [2,8- 3H]adenosine. The results demonstrated that benzamide riboside produced 2- and 3-fold more of NAD analogue (BAD) than tiazofurin and selenazofurin did. To elucidate the effects of the three compounds on other NAD-utilizing enzymes, the inhibitory activities of purified benzamide adenine dinucleotide (BAD), thiazole-4-carboxamide adenine dinucleotide (TAD) and selenazole-4-carboxamide adenine dinucleotide (SAD) were studied in commercially available purified preparations of lactate dehydrogynase, glutamate dehydrogenase and malate dehydrogenase. TAD and SAD did not inhibit these three dehydrogenases. Although BAD did not influence lactate and glutamate dehydrogenases, it selectively inhibited 50% of malate dehydrogenase activity at a 3.2 μM concentration. These studies demonstrate similarities and differences in the biochemical actions of the three C-nucleosides, even though they share similar mechanisms of action.

Cytotoxicity and characterization of an active metabolite of benzamide riboside, a novel inhibitor of IMP dehydrogenase.

Benzamide riboside exhibits significant cytotoxicity against a variety of human tumor cells in culture. On the basis of metabolic studies, the primary target of this drug's action appears to be IMP dehydrogenase (IMPDH). Incubation of human myelogenous leukemia K562 cells with an IC50 concentration of benzamide riboside resulted in an expansion of IMP pools (5.9-fold), with a parallel reduction in the concentration of GMP (90%), GDP (63%), GTP (55%) and dGTP (40%). On kinetic grounds, it was deduced that benzamide riboside (whose Ki versus IMPDH is 6.4 mM, while that of its 5'-monophosphate is 3.9 mM) or its 5'-monophosphate were unlikely to be responsible for inhibition of this target enzyme, IMPDH, since only micromolar concentrations of benzamide riboside were needed to exert potent inhibition of tumor-cell growth. Studies on the metabolism of this C-nucleoside have revealed the presence of a new peak eluting in the nucleoside diphosphate area on HPLC. Treatment of this peak with venom phosphodiesterase degraded it and concurrently nullified its inhibitory activity versus IMPDH; alkaline phosphatase, on the other hand, totally failed to digest the anabolite. These results suggest that the metabolite in question is the phosphodiester, benzamide adenine dinucleotide (BAD). Evidence that the inhibitor was an analog of NAD, wherein the nicotinamide moiety has been replaced by benzamide, was provided by both NMR and mass spectrometric analysis and confirmed by enzymatic synthesis. Further insight into the nature of the active principle was obtained from kinetic studies, which established that BAD competitively inhibited NAD utilization by partially purified IMPDH from K562 cells with a Ki of 0.118 microM. In concert, these studies establish that benzamide riboside exhibits potent antiproliferative activity by inhibiting IMPDH through BAD.

Purification and characterization of NF-R2 that regulates the expression of the human multidrug resistance (MDR1) gene.

NF‐R2 is a DNA‐binding protein that interacts with the MDR1 gene proximal promoter sequence. We previously reported that NF‐R2 binds within the promoter's — 126 and — 102 regions, which contain the ATTCAGTCA motif. In the present study, we have purified NF‐R2 from the nuclear extract of K562/ADM cells, a multidrug‐resistant cell line derived from human myelogenous leukemia K562 cells, using sequential chromatography on Sephacryl S‐300, DEAE‐Sepharose, heparin‐Sepharose and a DNA affinity column consisting of a repetitive synthetic ATTCAGTCA motif coupled to Sepharose. NF‐R2 runs as a single protein of 75 kDa on SDS‐PAGE (sodium dodecyl sulfate‐polyacrylamide gel electrophoresis). CAT (chloramphenicol acetyltransferase) expression assay and gel mobility shift competition assay with mutated promoters revealed that the ATTCAGTCA motif is a positive regulatory element of MDR1 gene and that the motif is important for NF‐R2 binding. These results suggest that NF‐R2 may be involved in the positive regulation of the MDR1 gene transcription.

Cytotoxicity of a new IMP dehydrogenase inhibitor, benzamide riboside, to human myelogenous leukemia K562 cells

COMPARE computer program suggested that benzamide riboside, BR, 3-(1-deoxy-beta-D-ribofuranosyl)benzamide, should have a similar mechanism of action as that of tiazofurin, an inhibitor of IMP dehydrogenase (IMPDH). This hypothesis was tested in K562 cells in culture. BR was cytotoxic to K562 cells with an IC50 of 2 microM. Incubation of K562 cells with BR resulted in a significant decrease in GMP and GTP levels with a concurrent increase in IMP pools, and with a significant inhibition of IMPDH activity. However, 290-fold higher BR concentration was needed to demonstrate in vitro inhibition of IMPDH activity, suggesting that the agent may require metabolism to exert its action. These results provide evidence that BR is a new inhibitor of IMPDH. This investigation should be helpful to design new analogues having activity against IMPDH.

Reversal of multidrug resistance by an immunosuppressive agent FK-506.

FK-506, a novel immunosuppressive agent, was examined for its reversing effect on multidrug-resistant tumor cells. FK-506 at 3 microM completely reversed the resistance against vincristine (VCR) in vitro in VCR-resistant mouse leukemia P388 cells (P388/VCR). FK-506 also enhanced the cytotoxicity of VCR in Adriamycin(ADM)-resistant human ovarian cancer A2780 cells (AD10) and ADM-resistant human myelogenous leukemia K562 cells (K562/ADM) in vitro. FK-506 was also effective in modulating sensitivity to ADM in AD10 cells in vitro. FK-506 enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice. When 20 mg/kg FK-506 was combined with 200 micrograms/kg VCR, a T/C value of 151% was obtained. Under the protocol used in this study, FK-506 was more potent than cyclosporin A (CsA) and verapamil. FK-506 inhibited [3H]azidopine binding to P-glycoprotein efficiently. The binding of VCR to K562/ADM plasma membrane was inhibited by FK-506 as effectively as by CsA. Moreover, the accumulation of VCR in AD10 cells was increased by FK-506 as efficiently as that of CsA and verapamil. These results indicate that FK-506 directly interacts with P-glycoprotein like CsA and verapamil, inhibits the active efflux of vincristine from resistant cells, increases the vincristine accumulation in resistant cells, and thus overcomes multidrug resistance in vitro and in vivo.

Cytotoxicity of tiazofurin and its arabinose and xylose analogues in K562 cells

2-beta-D-Arabinofuranosylthiazole-4-carboxamide and 2-beta-D-xylofuranosyl-thiazole-4-carboxamide are sugar modified analogues of tiazofurin, a C-glycosyl nucleoside which after anabolism to the dinucleotide, TAD (thiazole-4-carboxamide adenine dinucleotide), exhibits antitumor activity. However, ara-T and xylo-T did not exhibit cytotoxicity. Compared to tiazofurin, only 12.5% of the ara-T and 8.8% of the xylo-T were metabolized to TAD derivatives by human myelogenous leukemia K562 cells. This was reflected in the finding that guanylate pools were not depressed after treatment with either tiazofurin derivative. These results provide evidence that the ribose moiety is essential for the metabolism and cytotoxicity of tiazofurin. This investigation should be helpful in the design of new analogues of tiazofurin for future clinical trials.

Determination of NAD pyrophosphorylase activity in biological samples

A sensitive and simple method was developed for the accurate measurement of NAD pyrophosphorylase (NMN adenylyltransferase; EC 2.7.7.1) activity in biological samples. The reaction product of [4- 3H]NAD was separated from the substrates [4- 3H]NMN and ATP by HPLC. Under the standardized conditions of the assay, the enzyme activity in human chronic myelogenous leukemia K562 cells was found mainly in the nucleus (97%) with a sp act of 183.5 ± 3.5 nmol/h/mg protein. The K m 's for substrates NMN and ATP were 0.11 ± 0.01 m m and 0.55 ± 0.04 m m, respectively. This technique is highly reproducible with a 5% variation (SD) in five separate determinations. The lowest number of cells used for this enzyme assay was 41,000 with a protein content of 4 μg. The range of NAD produced during the assay was 2 to 200 μ m. NAD pyrophosphorylase activities in the mononuclear cells of leukemic patients, human ovarian carcinoma cells, and rat liver were assayed.

ATP/Mg2+-dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells.

To study the mechanism of active drug efflux in multidrug-resistant cells, the interaction between [3H] vincristine (VCR) and plasma membrane prepared from an adriamycin (ADM)-resistant variant (K562/ADM) of human myelogenous leukemia K562 cells was examined by filtration method. [3H]VCR bound to the plasma membrane prepared from K562/ADM cells, but not from parental K562 cells, depending on the concentrations of ATP and Mg2+. Adenosine 5'-O-(3-thio)triphosphate was not effective in the binding of [3H]VCR, indicating that ATP hydrolysis is required for this binding. Dissociation constant (Kd) of VCR binding was 0.24 +/- 0.04 microM in the presence of 3 mM ATP. In the absence of ATP, specific binding of VCR to K562/ADM membrane was also observed; however, the affinity (Kd = 9.7 +/- 3.1 microM) was 40 times lower than that observed in the presence of ATP. The high affinity VCR binding to K562/ADM membrane was dependent on temperature. The bound [3H]VCR molecules were rapidly released by unlabeled VCR added to the reaction mixture at 25 degrees C. The high affinity binding of [3H]VCR to K562/ADM membrane was inhibited by VCR, vinblastine, actinomycin D, and ADM, to which K562/ADM cells exhibit cross-resistance, whereas 5-fluorouracil and camptothecin, to which K562/ADM cells are equally sensitive as K562 cells, did not inhibit the [3H]VCR binding. Furthermore, verapamil and other agents, which are known to circumvent drug resistance by inhibiting the active efflux of antitumor agents from resistant cells, could also inhibit the high affinity [3H]VCR binding. These results indicate that ATP/Mg2+-dependent high affinity VCR binding to the membrane of resistant cells closely correlates with the active drug efflux of this resistant cell line.

Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. 170- to 180-kilodalton membrane glycoprotein is an ATPase.

170-180-kDa membrane glycoprotein (P-glycoprotein) associated with multidrug resistance is involved in drug transport mechanisms across the plasma membrane of resistant cells. From sequence analysis of cDNAs of the P-glycoprotein gene, it is postulated that the active drug-efflux pump function may be attributable to the protein. However, purification of the P-glycoprotein while preserving its enzymatic activity has not been reported. In this study, we have purified the P-glycoprotein from the human myelogenous leukemia K562 cell line resistant to adriamycin (K562/ADM) by means of one-step immunoaffinity chromatography using a monoclonal antibody against P-glycoprotein. The procedure was simple and efficiently yielded an electrophoretically homogeneous P-glycoprotein sample. By solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified P-glycoprotein was found to have ATPase activity. This ATP hydrolysis may be coupled with the active efflux of anticancer drugs across the plasma membrane of multidrug-resistant cells.

Functional role for the 170- to 180-kDa glycoprotein specific to drug-resistant tumor cells as revealed by monoclonal antibodies.

An overexpression of the plasma membrane glycoprotein of relative molecular size 170-180 kDa is consistently found in different multidrug-resistant human and animal cell lines, although the functional role of the protein in multidrug resistance is not known. Two monoclonal antibodies that interfere with biochemical functions were generated against the human myelogenous leukemia K-562 cells resistant to adriamycin (K-562/ADM). These antibodies, designated MRK16 and MRK17, are specifically reactive to K-562/ADM and a human ovarian cancer cell line resistant to adriamycin (2780AD). MRK16 modulated vincristine and actinomycin D transport in the resistant cells, while MRK17 specifically inhibited the growth of the resistant cells. Both antibodies recognized the 170- to 180-kDa glycoprotein. These data indicate that the 170- to 180-kDa glycoprotein is involved, directly or indirectly, in the drug transport mechanisms and the proliferation of multidrug-resistant tumor cell lines.

Reproducible Metastatic Growth of K-562 Human Myelogenous Leukemia Cells in Nude Mice<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

A reproducible metastatic growth of K-562 human myelogenous leukemia cells occurred in immunodeficient athymic (nude) mice. Although previous studies have shown that K-562 cells grow as local subcutaneous myelosarcomas which continuously release leukemia cells into the systemic circulation in adult mice, metastases were not observed. However, the subcutaneous "priming" of newborn nude mice resulted in the metastatic proliferation of leukemia cells in the lungs, kidneys, brain, and lymph nodes. Three sc injections of 5 X 10(6) K-562 cells on days 1, 7, and 14 of life produced metastases in 51% of the mice. When the initial series of injections was followed by iv injections on days 35 and 42, the incidence of metastases increased to 67%. Karyotypes demonstrated that the tumor cells retained the same human chromosome markers as those in the human patient and tissue culture. These procedures may provide a model for study of the mechanisms of metastases and for chemotherapeutic and immunotherapeutic trials against metastases of neoplasms of human origin.