Mass spectrometric analysis of affinity-purified proteasomes from the human myelogenous leukemia K562 cell line

Proteasomes carry out regulated proteolysis of most proteins in a cell and thereby play a crucial role in the regulation of various cellular processes. Determination of the subunit composition and posttranslational modifications of proteasomes is one of the important stages in understanding of proteasomes functions in the cell and mechanisms of their regulation. To solve this problem a strategy of affinity purification of proteasomes with the subsequent mass spectrometric analysis has been implemented, using human myelogenous leukemia cells. Proteasomes have been purified from the stable K562 cell line expressing β7 (PSMB4) subunit of the 20S proteasome tagged with C-terminal HTBH peptide containing two His₆ fragments, the specific site of cleavage by Tobacco Etch Virus (TEV) protease, and signal sequence for biotinylation in vivo, using method of noncovalent binding through formation of biotin complex with streptavidin with the subsequent elution with TEV protease. All known subunits of the 26S proteasome, as well as PA200 and PA28γ regulators have been identified using MALDI FT-ICR mass spectrometry. We have demonstrated that the heat shock proteins, components of the ubiquitin-proteasome system, and some cytoskeleton proteins are associated with proteasomes. A number of new sites of phosphorylation, ubiquitination, and N-terminal modification have been found for 16 proteasome subunits. The presented mass spectrometric analysis will be useful for the further proteomic studies of proteasomes under cellular stress.     

Phenylobacterium zucineum sp. nov., a facultative intracellular bacterium isolated from a human erythroleukemia cell line K562

A bacterial strain HLK1(T) was isolated from the human erythroleukemia cell line K562. This bacterium is a Gram-negative rod, motile with a polar flagellum. It is strictly aerobic, nonfermentative, and oxidase and catalase positive. Its optimal growth occurs at 37 degrees C at pH between 6.5 and 7.5. Phylogenetically, although it shares 98% similarity with the 16S rRNA of Phenylobacterium lituiforme, the DNA-DNA hybridization value between the two species is only 43%. HLK1(T) has a DNA G+C content of 71.2+/-0.2 mol%. It is a facultative intracellular organism and may have pathogenic relevance with humans and mammals. On the basis of the phylogenetic and phenotypic characterization, strain HLK1(T) is proposed to be classified in the genus Phenylobacterium, as P. zucineum sp. nov. The type strain is HLK1(T) (=CGMCC 1.3786(T), DSM=18354).     

Embryonic and fetal hemoglobin synthesis in K562 cell line

K562 cell line was grown in liquid suspension and in plasma clot cultures. Morphological studies revealed the presence of a minority of cells, which were identified as erythroblasts. However, the majority of the cells remained unidentified. Biochemical studies confirmed the synthesis of hemoglobin by K562 cells. The pattern of hemoglobin (Hb) production was of the embryonic type, with the presence of small amount of fetal Hb. The addition of several inducers, like Epo and butyrate, was unable to modify the pattern of Hb production of K562. In contrast, the addition of hemin increased the synthesis of Hb and stimulated the synthesis of fetal Hb and probably adult Hb.     

Properties of endoribonuclease activity of proteasomes from K562 cells. II. Analysis of specific mRNA nucleolysis by proteasomes

The ability of 26S proteasomes from the human proerythroleukaemic cell line K562 to degrade high-molecular-weight cytoplasmic RNAs, particularly specific messenger RNA, has been detected. The addition of hemin to K562 cells in the culture media leads to redistribution of proteasomes and their migration mainly to the cytoplasm. The human wild type p53 gene mRNA was shown to be specifically nucleolized by proteasomes. These particles displayed endoribonuclease activity towards mRNA for Renilla sp. luciferase. Proteasomes also specifically degraded Alu-containing mRNAs. A supposition is made about the involvement of proteasomes in stability control of specific RNA groups.     

Induction of hemoglobin synthesis in original K 562 cell line

Cells of the original human myelogenous leukemia cell line, K 562 were induced to synthesize hemoglobin by incubation with 0.05 mM hemin. Analysis by isoelectric focusing indicated major bands in the region of hemoglobin F, Bart's hemoglobin, and embryonics. Cytochemical analysis by the Lepehene peroxidase reaction for hemoglobin indicated an heterogeneous population of cells with respect to hemoglobin induction. Approximately 50% of the cells were positive for hemoglobin. The benzidine-positive material was present as granules in the region of the Golgi apparatus of large blasts with undifferentiated nuclei. There was no indication of normal maturation or of terminal differentiation into reticulocytes or erythrocytes.     

Characterization of insulin binding to the erythroleukemia cell line K 562

The K 562 is a transformed human erythroid stemcell and is used as a target cell for NK-T-cells. In this study the presence of insulin receptors in K 562 is established.The best binding and negative cooperativity was found in the two Hepes containing buffers whereas no cooperativity was obtained in the Krebs-Ringer buffer. The calculated affinity constants and receptor number per cell varied according to the buffer. Preincubation with insulin caused a down-regulation of the insulin binding capacity. 10 ng/ml caused a lowering of the affinity, with an unchanged number of receptors. 100 ng/ml caused a decrease in receptor number with unchanged affinity. These results were found in both Hepes and Krebs-Ringer phosphate buffer. IGF-I shows cross-reactivity with the insulin receptor, with a potency of 12 and 100 times less than insulin in Krebs-Ringer phosphate buffer and G-buffer respectively. However, no specific IGF-I receptors were found.The presence of receptors on K 562 cells suggests a biological role for insulin. The different results in the different buffers, indicate that a buffer containing Hepes and/or Tris, is required to expose negative cooperativity and make the receptors more accessible to insulin.     

The binding of insulin-like growth factor II to the erythroleukemia cell line K562

The erythroleukemia cell line K562 was previously shown to have specific binding sites for insulin but not for insulin-like growth factor I (IGF-I). In this study the presence of specific receptors for insulin-like growth factor II (IGFqI) is established. Scatchard analysis of the competition curve for IGF-II disclosed a non-cooperative binding kinetic with a calculated affinity constant of 2.4×108 M^–1 and a receptor number of 4.8×l0⁴ sites/cell. IGF-I displayed 10% crossreactivity over the IGF-II receptor but insulin did not crossreact at all. Instead insulin, present in high concentrations, enhanced the binding of IGF-II. The presence of IGF II but not IGF-I receptors makes t h e K562 cell line suitable for studying properties of the type-2 receptor.     

Nitric oxide levels during erythroid differentiation in K562 cell line

Nitric oxide (NO) is endogenous mediator of numerous physiological processes that range from regulation cardiovascular function and neurotransmission to antipathogenic and tumoricidal responses. This study was designed to investigate the possible role of NO during erythroid differentiation in K562 erythroleukemia cells. The chronic myelogenous leukemia (K562) cell line can be triggered in culture to differentiate along the erythrocytic pathway, in response to a variety of stimulatory agents. In this study, K562 cells were induced to synthesize hemoglobin by hemin. We investigated NOx (nitrate+nitrite) levels in uninduced (control) and hemin-induced K562 cell lysates during erythroid differentiation. Our results showed that NO levels decreased significantly on fourth and sixth day both in hemin-induced and control cells; the decrease was, however, more in hemin-induced group than in control group.     

Terminal differentiation of human erythroleukemia cell line K562 induced by aphidicolin

To analyze the relationship between differentiation and DNA replication, the effect of aphidicolin, a specific inhibitor for DNA polymerase alpha, was measured with respect to erythroid differentiation and activities of DNA polymerases alpha, beta, and gamma. Five micromolar aphidicolin completely blocked the growth of K562 cells and caused 80% of cells to become hemoglobin positive after 5 days exposure. The cessation of K562 cell growth induced by aphidicolin was irreversible, whereas the inhibition of HeLa cell growth was completely reversible. The enzyme activity of DNA polymerase alpha of K562 cells showed a 50-110% increase with aphidicolin treatment as compared to control K562 cells; activities of DNA polymerases beta and gamma were not affected. These features sharply contrasted with the erythroid induction of the same cells by hemin, where cell growth was not suppressed and DNA polymerase alpha was not increased but rather decreased. The enzyme activity of DNA polymerase alpha remained high even after removal of aphidicolin from the culture medium. These results suggest that treatment with aphidicolin might induce an accumulation of protein factors for replication and/or differentiation, causing rapid cell differentiation of cells without cell division.     

Partial purification of an IGF-II receptor/binding protein from the erythroleukemia cell line K562

We previously reported that insulin-like growth factor II (IGF-11) stimulated clonal growth of an erythroleukemia cell line, K562, in semi-solid agar, an effect not mimicked by insulin-like growth factor I (IGF-1), as IGF-I receptors are generally not expressed in this cell line. Affinity crosslinking of intact K562 cells with ¹²⁵I-IGF-II revealed that the labeled hormone predominantly bound to a protein with a molecular weight of approximately 75 K. We report here the partial purification of the 75 K IGF-II binding protein from K562 cells. Triton X-100-solubilized K562 cells were subjected to Sephacryl-400, followed by Sephacryl-200 chromatography. Fractions of interest were collected and applied to a Sepharose-IGF-II column or an immunoaffinity column. The immuno-affinity column was prepared using an antiserum against placental membrane-derived material eluted from the Sephacryl-400 column in the elution volume, corresponding to the IGF-II binding protein from K562 cells. An affi-gel 10 affinity column, prepared with a protein A purified IgG fraction of this antiserum (antibody-29), retarded proteins showing binding specificity for IGF-II, with apparent molecular weights of 76 K, 87 K, and 70 K under reducing conditions. These protein bands were similar to the proteins retarded in the IGF-II affinity column, when evaluated by affinity crosslinking and SDS-PAGE. Fractionation of the purified material from the antibody-29 affinity column on Superose 12 revealed 6 protein peaks. Affinity crosslinking of the peak fractions from FPLC resulted in single bands with a molecular weight of 75 K under reducing conditions with variable specificity for IGF-II.     

Immunochemical detection of cell cycle synchronization in a human erythroleukemia cell line, K562

Cells of the human erythroleukemic line K562 can be induced by manipulation of culture conditions to arrest within the G1 phase of the cell cycle, and subsequently to enter S phase synchronously after release from G1. Cell cultures subjected to serum deprivation and hydroxyurea (HU) treatment demonstrated less than 5% of the cells to be in S phase. Four hours after release from HU, 63% of the cells were in S phase, as detected by immunofluorescent staining. This protocol offers a method for synchronization of K562 cells at the G1/S border and a technique for detection of S-phase cells without the use of radioisotopes or flow cytometry instrumentation.     

Volume-activated taurine permeability in cells of the human erythroleukemic cell line K562

The effects of hypotonic shock on cell volume, taurine influx and efflux were examined in the human erythroleukemic cell line K562. Cells exposed to hypotonic solutions exhibited a regulatory volume decrease (RVD) following rapid increases in cell volume. Cell swelling was associated with a increased taurine influx and efflux. The volume-activated taurine pathway was Na⁺-independent, and increased in parallel with increasing cell volume. The chloride channel blocker, 2,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), completely blocked the volume-activated taurine influx and efflux, while [dihydroin-denyl]oxy]alkanoic acids (DIOA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an anion exchanger and anion channel blocker, respectively, also inhibited significantly. These results suggest that taurine transport is increased in response to hypotonic stress, which may be mediated via a volume-activated, DCDPC-sensitive anion channel. © 1996 Wiley-Liss, Inc.     

Comparison of different methods for erythroid differentiation in the K562 cell line

Objective

To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis.

Results

Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001).

Conclusions

Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.

     

Detection and analysis of extra- and intracellular deoxyribonuclease activities in Lactobacillus plantarum

Extracellular endodeoxyribonuclease activities have been detected in culture supernatant fluids of several Lactobacillus plantarum strains. In the case of Lact. plantarum HER 1325 at least, the extracellular activity is accompanied by a cytoplasmic restriction endonuclease. Among the secreted nucleases, the greatest activity was from Lact. plantarum ATCC 10241. This strain secretes an enzyme, with a molecular mass in the range 10–40 kDa, that cuts double-stranded DNA in a sequence-independent way by initially introducing single-strand nicks in supercoiled molecules, followed by linearization and complete degradation of the substrate.     

Transferrin binding to K562 cell line : Effect of heme and sodium butyrate induction

Experiments demonstrating the existence of receptors for iron-saturated transferrin on K562 cells are described. Binding of ¹²⁵I-labelled transferrin is rapid, saturable and reversible, and can be specifically inhibited by unlabelled transferrin, but not by other proteins. The number of receptors determined by Scatchard analysis significantly decreased when K562 cells moved from the exponential to the quiescent phase of growth. Induction by hemin or sodium butyrate resulted in a marked reduction of transferrin binding. This phenomenon was due entirely to reduction in the number of receptors and was without effect on the affinity of interaction. The effect of butyrate and hemin on the number of transferrin receptors in other hematopoietic cell lines was investigated. Butyrate on the various cell lines was variable in its effect, whereas hemin constantly elicited a significant reduction in the number of transferrin receptors.     

SHIP2 overexpression strongly reduces the proliferation rate of K562 erythroleukemia cell line

SHIP2 belongs to the inositol 5-phosphatase family and is characterized by a phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) 5-phosphatase activity. Evidence based on mice lacking the SHIP2 gene has demonstrated its predominant role in the control of insulin sensitivity. However, SHIP2 expression in both hematopoietic and non-hematopoietic cells suggests additional functions. SHIP2 was previously identified in chronic myelogenous progenitor cells, in which its constitutive tyrosine phosphorylation was reported by Wisniewski et al., [Blood 93 (1999) 2707-2720]. Here, we further investigated the function of SHIP2 in this hematopoietic and malignant context. A detailed analysis of the substrate specificity of SHIP2 indicated that this phosphatase is primarily directed towards PI(3,4,5)P(3) both in vitro and in K562 chronic myeloid leukemia cells. The SHIP2-mediated decrease in PI(3,4,5)P(3) levels and increase in phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) was accompanied by a reduction of cell proliferation, characterized by an accumulation of the cells in the G2/M phase of the cell cycle. Thus, in addition to its role in the control of insulin sensitivity, SHIP2 may also play a role in cell proliferation, at least in chronic myelogenous progenitor cells.     

A critical intracellular concentration of fully reduced non-methylated folate polyglutamates prevents macrocytosis and diminished growth rate of human cell line K562 in culture.

Growth rate of human leukaemic cell line K562 was independent of intracellular folate concentration when this was greater than 1.5 microM. When intracellular folate concentration was less than 1.5 microM, the rate of growth was proportional to the logarithm of intracellular concentration of non-methylated fully reduced folates, but not to the logarithm of the intracellular concentration of N5-methyltetrahydropteroylglutamate. Intracellular folate concentration sufficient to support an optimal growth rate was maintained by either DL-N5-formyltetrahydropteroylglutamate or DL-N5-methyltetrahydropteroylglutamate at a 100-fold lower concentration than pteroylglutamate. Addition of hypoxanthine to culture medium partially restored growth of folate-depleted cells: thymidine had no effect on growth rate either alone or in combination with thymidine. Folate-depleted cells with diminished growth rate were larger than replete cells, but did not have megaloblastic morphology. The mitotic index was not decreased in cultures with diminished growth rate. The rate of growth and cell size of K562 cells is thus dependent on a critical intracellular concentration of non-methylated tetrahydrofolates, which may be maintained by different concentrations of either reduced folates or pteroylglutamate.