Incubation Of K562 Cells Research Articles

Anti-Proliferative and Apoptotic Effects of Beta-Ionone in Human Leukemia Cell Line K562

Background: Beta-ionone is an aroma compound found in the Rosaceae family. Some evidence supported that beta-ionone has a great potential for cancer prevention. To date, the anti-proliferative and apoptotic effects of beta-ionone in human leukemia cell line K562 were not studied. Objectives: Hence, we investigated whether beta-ionone could inhibit cell growth and induce apoptosis in the K562 cells. Materials and Methods: In this experimental study, human leukemia cell line K562 was cultured and anti-proliferation effect of beta-ionone with different doses (25 - 400 µm) at different times (24 - 96 hours) on treated cells was evaluated by the MTT assay. To determine apoptosis rate, the Hoechst 33342 staining and flow cytometry was performed. Results: The MTT assay showed that beta-ionone inhibited proliferation of K562 cells in a dose-dependent manner significantly (P = 0.0008). Moreover, the increased apoptotic rate was found after incubation of K562 cells with 200 µm beta-ionone. The Hoechst staining and flow cytometry analysis indicated that beta-ionone could increase apoptosis of K562 cells in a dose-dependent manner. Conclusions: The results demonstrated that beta-ionone has anti-proliferative and apoptotic effects on K562 cells, and in the future may be used in the treatment of some leukemia sub-types.

Phloretin increases the anti-tumor efficacy of intratumorally delivered heat-shock protein 70kDa (HSP70) in a murine model of melanoma.

Recombinant HSP70 chaperone exerts a profound anticancer effect when administered intratumorally. This action is based on the ability of HSP70 to penetrate tumor cells and extract its endogenous homolog. To enhance the efficacy of HSP70 cycling, we employed phloretin, a flavonoid that enhances the pore-forming activity of the chaperone on artificial membranes. Phloretin increased the efficacy of HSP70 penetration in B16 mouse melanoma cells and K-562 human erythroblasts; this was accompanied with increased transport of the endogenous HSP70 to the plasma membrane. Importantly, treatment with HSP70 combined with phloretin led to the elevation of cell sensitivity to cytotoxic lymphocytes by 16-18% compared to treatment with the chaperone alone. The incubation of K-562 cells with biotinylated HSP70 and phloretin increased the amount of the chaperone released from cells, suggesting that chaperone cycling could trigger a specific anti-tumor response. We studied the effect of the combination of HSP70 and phloretin using B16 melanoma and a novel method of HSP70-gel application. We found that the addition of phloretin to the gel reduced tumor weight almost fivefold compared with untreated mice, while the life span of the animals extended from 25 to 39days. The increased survival was corroborated by the activation of innate and adaptive immunity; interestingly, HSP70 was more active in induction of CD8+cell-mediated toxicity and γIFN production while phloretin contributed largely to the CD56+ cell response. In conclusion, the combination of HSP70 with phloretin could be a novel treatment for efficient immunotherapy of intractable cancers such as skin melanoma.

Cytotoxic action of bisabololoxide A of German chamomile on human leukemia K562 cells in combination with 5-fluorouracil

German chamomile (Matricaria recutita L.) is a popular ingredient in herbal teas. In previous study, micromolar bisabololoxide A, one of main constituents in German chamomile, exerted cytotoxic action on rat thymocyte, a normal non-proliferative cell. This result prompted us to study the effect of bisabololoxide A on proliferative cancer cells and to seek the possibility of its use with 5-fluorouracil, an anticancer agent. In this study, the effect of micromolar bisabololoxide A on human leukemia K562 cells was cytometrically examined. Although the incubation of K562 cells with 10 μM bisabololoxide A for 72h did not significantly increase the percentage populations of dead cells and shrunken cells, the inhibitory action on the growth was obviously observed. It was not the case for the concentrations of less than 5 μM. The threshold concentration of bisabololoxide A to exert the cytotoxic action on K562 cells was ascertained to be 5-10 μM. Bisabololoxide A at 5-10 μM did not exert cytotoxic action on normal non-proliferative cells (rat thymocytes) in our previous study. Since the antiproliferative action of micromolar bisabololoxide A on cancerous cells was expected to be beneficial to cancer treatment, the modification of antiproliferative action of 5-fluorouracil (3-30 μM) by bisabololoxide A was studied. The combination of 5-fluorouracil and bisabololoxide further inhibited the growth of K562 cells although the additive inhibition of growth by bisabololoxide A became smaller as the concentration of 5-fluorouracil increased. Therefore, it is suggested that the simultaneous application of German chamomile containing bisabololoxide A may reduce the dose of 5-fluorouracil.

Study of diosgenin-induced apoptosis kinetics in K562 cells by Sedimentation Field Flow Fractionation

Recently, the use of SdFFF in cancer research has been studied in order to better understand major phenomena implicated in cancer development and therapy: apoptosis and differentiation. In this report, we used SdFFF as a monitoring and cell separation tool to study the kinetics of apoptosis. Incubation of K562 cells with diosgenin, used as cellular model, led to a surprising apoptotic process occurring in two phases (after 24 and 48 h incubation), associated with specific p-ERK expression. Based on the capacity to sort apoptotic cells, results showed that SdFFF cell separation was an effective analytical tool to obtain different subpopulations regardless of the kinetics and extent of apoptosis. Results also showed that, after proper biological calibration of elution profiles, SdFFF can be used to monitor either the induction or the kinetics of a biological event.

Glutathione level regulates HNE-induced genotoxicity in human erythroleukemia cells

4-Hydroxy- trans-2-nonenal (HNE) is one of the most abundant and toxic lipid aldehydes formed during lipid peroxidation by reactive oxygen species. We have investigated the genotoxic effects of HNE and its regulation by cellular glutathione (GSH) levels in human erythroleukemia (K562) cells. Incubation of K562 cells with HNE (5–10 μM) significantly elicited a 3- to 5-fold increased DNA damage in a time- and dose-dependent manner as measured by comet assay. Depletion of GSH in cells by l-buthionine-[S,R]-sulfoximine (BSO) significantly increased HNE-induced DNA damage, whereas supplementation of GSH by incubating the cells with GSH-ethyl ester significantly decreased HNE-induced genotoxicity. Further, overexpression of mGSTA4-4, a HNE-detoxifying GST isozyme, significantly prevented HNE-induced DNA damage in cells, and ablation of GSTA4-4 and aldose reductase with respective siRNAs further augmented HNE-induced DNA damage. These results suggest that the genotoxicity of HNE is highly dependent on cellular GSH/GST/AR levels and favorable modulation of the aldehyde detoxification system may help in controlling the oxidative stress-induced complications.

Endocytotic Internalization as a Crucial Factor for the Cytotoxicity of Ribonucleases

The cytotoxic action of ribonucleases (RNases) requires the interaction of the enzyme with the cellular membrane, its internalization, translocation to the cytosol, and the degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the evasion from the intracellular ribonuclease inhibitor (RI) has not yet been fully elucidated. As cytosolic internalization is indispensable for the cytotoxicity of extracellular ribonucleases, we investigated the extent of cytosolic internalization of a cytotoxic, RI-evasive RNase A variant (G88R-RNase A) and of various similarly cytotoxic but RI-sensitive RNase A tandem enzyme variants in comparison to the internalization of the non-cytotoxic and RI-sensitive RNase A. After incubation of K-562 cells with the RNase A variants for 36 h, the internalized amount of RNases was analyzed by rapid cell disruption followed by subcellular fractionation and semiquantitative immunoblotting. The data indicate that an enhanced cellular uptake and an increased entry of the RNases into the cytosol can outweigh the abolishment of catalytic activity by RI. As all RNase A variants proved to be resistant to the proteases present in the different subcellular fractions for more than 100 h, our results suggest that the cytotoxic potency of RNases is determined by an efficient internalization into the cytosol.

A CELL SURFACE TISSUE TRANSGLUTAMINASE IS INVOLVED IN RECOGNITION OF GLIADIN PEPTIDES BY K 562 CELLS

Aim: Several studies have been demonstated that the agglutination activity of human chronic myelogenous leukemia K562 cells by prolamines peptictryptic digest (PT) is higly correlated with the toxicity of these prolamines in coeliac disease. The mechanism of the agglutination activity is unknown, but the presence of a binding site on cell surface of K562 cells has been supposed. In the latest years, a cell surface tissue transglutaminase has been identified in CD8 + T cell, in monocytes and in K562 cells. Tissue transglutaminase is known to play a pivotal role in pathogenesis of coeliac disease, binding the gliadin peptides in the small bowel mucosa of these patients. We hypothesize that the cell surface transglutaminase could have a role in the agglutination activity of K562 cells. Methods: K562 cells were grown in RPMI medium. For the agglutination test, cells were harvested, washed and resuspended in PBS at the final concentration of 108cells/ml. The agglutination test were carried out as follow: in each well of a 96 well plate 25 μl of PBS and 25 μl of cells were added. Then, mAb anti tTG CUB 7402 (Neomarker, USA) 1 μl of a solution at the concentration of 2 μg/ml and 25 μl of a 0,5 mg/ml PT solution were added. Finally, an incubation for 30 min occoured before evaluating the agglutination by optical microscope. Controls were performed with PT only and with mAB anti rabbit IgG. Results: The incubation of K562 cells with PT digest resulted in a massive agglutinationof the cells. No agglutination occoured when the mAb anti tTG was added prior to the PT digest. Summary: The ability of the mAb anti tTG of preventing the agglutination of K562 by gliadin peptides indicates that a cell surface tissue transglutaminase acts as binding site for the gliadin peptides. Conclusion: The results of these study suggest that tissue transglutaminase could play a role in recognition of gliadin peptides by the cells in coeliac disease and it could be involved in starting other responses than the activation of the adaptive immune system in the mucosa of the coeliac disease patients.

Regulation of Equilibrative Nucleoside Uptake by Protein Kinase Inhibitors

For Abstract see ChemInform Abstract in Full Text.

Regulation of Equilibrative Nucleoside Uptake by Protein Kinase Inhibitors

The uptake of nucleosides and nucleoside analogs into human leukemia K562 cells is facilitated by the equilibrative transporters ENT1 and ENT2. Incubation of K562 cells with a variety of protein kinase inhibitors inhibited the transport of both uridine (ARA‐C) and cytidine (CPEC) analogs. These inhibitory effects were observed for a large number of kinase inhibitors including those against p38 MAPK, the EGF receptor kinase, protein kinase C, TOR and others. Thus these results suggest that the nucleoside transporters are unexpected targets for kinase inhibitors and may influence the design and application of combinatorial approaches of nucleoside analogs and kinase inhibitors in clinical applications.

Inhibition of nucleoside transport by protein kinase inhibitors.

Recently we reported that the pyridinylimidazole class of p38 mitogen-activated protein (MAP) kinase inhibitors potently inhibited the facilitated transport of nucleosides and nucleoside analogs in K562 cells. These compounds competed with the binding of nitrobenzylthioinosine (NBMPR) to K562 cells, consistent with inhibition of the NBMPR-sensitive equilibrative transporter (ENT1). In this study we examined a large number of additional protein kinase inhibitors for their effects on nucleoside transport. We find that incubation of K562 cells with tyrosine kinase inhibitors (AG825, AG1517, AG1478, STI-571), protein kinase C (PKC) inhibitors (staurosporine, GF 109203X, R0 31-8220, arcyriarubin A), cyclin-dependent kinase inhibitors (roscovitine, olomoucine, indirubin-3'-monoxime), or rapamycin resulted in a dose-dependent reduction of intracellular uptake of [3H]uridine. In contrast, neither the MAP kinase kinase inhibitors (U0126, PD 98059) nor the phosphatidyl inositol-3 kinase inhibitors (wortmannin, LY 294002) affected this process. Furthermore, both transient uptake and prolonged [3H]thymidine incorporation in K562 cells were inhibited by protein kinase inhibitors, inactive analogs of kinase inhibitors (R0 31-6045, SB202474), and NBMPR, independently of effects on cell proliferation as determined by MTT assay. These studies demonstrate that a wide variety of protein kinase inhibitors affect nucleoside uptake through selective inhibition of nucleoside transporters, independently of kinase inhibition.

Calcitonin receptor gene expression in K562 chronic myelogenous leukemic cells

BackgroundThe peptide hormone calcitonin (CT) can significantly effect the proliferation rate of CT receptor (CTR) positive human cancer cells. We wish to identify additional human cancers expressing CTRs and assay the effects of CT on their growth rates and signal transduction pathways.ResultsThe expression of the human calcitonin receptor (hCTR) gene in the chronic myelogenous leukemia cell line K562 was examined. RT-PCR on total RNA extracted from K562 cells detected the presence of hCTR mRNA. Further analysis demonstrated that multiple hCTR isoforms were present. Incubation of K562 cells with salmon calcitonin (sCT), but not amylin, caused an increase in intracellular levels of cAMP similar to that induced by forskolin treatment. We further demonstrated that butyrate induced erythroid differentiation of K562 cells caused a significant decrease in hCTR mRNA levels. However, phorbol myristate acetate (PMA) induced megakaryocytic differentiation of these cells had no significant effect on hCTR mRNA levels. We demonstrated that exposure to various concentrations of sCT had no effect on the cellular proliferation of K562 cells in vitro.ConclusionChronic myelogenous k562 cells express multiple CTR isoforms. However, CT does not effect K562 proliferation rates. It is likely that the small increase in intracellular levels of cAMP following CT treatment is not sufficient to interfere with cellular growth.

Inhibition of Nucleoside Transport by p38 MAPK Inhibitors

While investigating the ability of p38 MAPK to regulate cytarabine (Ara C)-dependent differentiation of erythroleukemia K562 cells, we observed effects that indicated that the imidazoline class of p38 MAPK inhibitors prevented nucleoside transport. Incubation of K562 cells with SB203580, SB203580-iodo, or SB202474, an analogue of SB203580 that does not inhibit p38 MAPK activity, inhibited the uptake of [3H]Ara C or [3H]uridine and the differentiation of K562 cells. Consistent with the effects of these compounds on the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter (ENT1), incubation with SB203580 or SB203580-iodo eliminated the binding of [3H]NBMPR to K562 cells or membranes isolated from human erythrocytes. Furthermore, using a uridine-dependent cell type (G9c), we observed that SB203580 or SB203580-iodo efficiently inhibited the salvage synthesis of pyrimidine nucleotides in vivo. Thus these studies demonstrate that the NBMPR-sensitive equilibrative nucleoside transporters are novel and unexpected targets for the p38 MAPK inhibitors at concentrations typically used to inhibit protein kinases.

Characteristic features of embryogenesis and oogenesis in the beetlesAdalia bipunctata L. andHarmonia axyridis Pall. in all-female families

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to non-specific lysis (non-restricted with respect to antigens of the major histocompatibility complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.

Exposure to sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristine-13-acetate alters sensitivity of K562 cells to nonspecific lysis by human or rat leukocytes

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to nonspecific lysis (nonrestricted with respect to antigens of the major histocompatibilty complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.

Cytotoxicity of an 125I-Labelled DNA Ligand

The subcellular distribution and cytotoxicity of a DNA-binding ligand [125I]-Hoechst 33258 following incubation of K562 cells with the drug was investigated. The ability of a radical scavenger, dimethyl sulphoxide, to protect cells from the 125I-decay induced cell death was also studied. Three different concentrations and specific activities of the drug were used to provide different ligand : DNA binding ratios. The results demonstrated a trend toward improved delivery of the ligand to the nucleus and to chromatin at higher ligand concentrations, with concomitant increased sensitivity to 125I-decay induced cytotoxicity and decreased protection by dimethyl sulphoxide. This correlation of radiobiological parameters with subcellular drug distribution is consistent with the classical dogma that attributes cytotoxicity to DNA double-stranded breakage in the vicinity of the site of decay, where the high LET nature of the damage confers minimal sensitivity to radical scavenging.

DNA alkylation and interstrand cross-linking by treosulfan.

The anti-tumour drug treosulfan (L-threitol 1,4-bismethanesulphonate, Ovastat) is a prodrug for epoxy compounds by converting non-enzymatically to L-diepoxybutane via the corresponding monoepoxide under physiological conditions. The present study supports the hypothesis that this conversion of treosulfan is required for cytotoxicity in vitro. DNA alkylation and interstrand cross-linking of plasmid DNA is observed after treosulfan treatment, but this is again produced via the epoxide species. Alkylation occurs at guanine bases with a sequence selectivity similar to other alkylating agents such as the nitrogen mustards. In treosulfan-treated K562 cells, cross-links form slowly, reaching a peak at approximately 24 h. Incubation of K562 cells with preformed epoxides shows faster and more efficient DNA cross-linking. © 1999 Cancer Research Campaign

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.

Induction of carbonic anhydrase I isozyme precedes the globin synthesis during erythropoiesis in K562 cells

Induction of carbonic anhydrase isozyme I (CA-I) by erythropoietin or hemin was investigated using erythroleukemia (K562) cells. Immunological estimation and purification of carbonic anhydrases showed that untreated K562 cells contained only carbonic anhydrase isozyme II(CA-II), while incubation of the cells with 2 units of erythropoietin (EP) per ml of the incubation medium or with 50 microM hemin resulted in the induction of CA-I. The purified CA-I induced in K562 cells was enzymatically and immunologically identical to that from mature erythrocytes. Flow cytometric analysis showed that incubation of K562 cells with EP as well as hemin induced CA-I at the 3rd h, while alpha-globin was detected at the 8th h. Northern blot analysis of CA-I mRNA using a cloned genomic DNA as a probe showed that mRNA of CA-I was induced by EP. These results suggest that induction of CA-I is regulated at the transcriptional level during developmental changes of erythroid cells, and that CA-I may play a physiologically important role during erythroid differentiation.

Glycosylation of CD45: carbohydrate composition and its role in acquisition of CD45RO and CD45RB T cell maturation‐related antigen specificities during biosynthesis

The contribution of oligosaccharides to the biochemical composition and antigen heterogeneity of the phosphotyrosine phosphatase CD45 glycoproteins has been studied on the K-562 erythroleukemic cell line. Treatment of immunoprecipitated CD45 glycoproteins with distinct exo- and endoglycosidases revealed the presence of highly sialylated O- and N-linked complex carbohydrates in the composition of mature CD45 glycoproteins. Incubation of K-562 cells with the N-glycosylation inhibitor tunicamycin blocked carbohydrate processing during biosynthesis of CD45 proteins, generating unglycosylated polypeptides similar in size to those resulting from digestion of CD45 proteins with a mixture of both N- and O-glycanases. Epitopes defining the T cell maturation related CD45R0 and CD45RB antigen specificities were present on the mature 180- and 190-kDa K-562 CD45 proteins, respectively. However, the CD45R0 and CD45RB epitopes were not detected on the high mannose biosynthetic CD45 precursors. Furthermore, treatment of CD45 proteins with O-glycanase or neuraminidase resulted in the loss of both CD45R0 and CD45RB epitopes, although reactivity of the anti-CD45R0 and anti-CD45RB mAb was not affected by mAb preincubation with either sialic acids or sialyllactose in solution. From these results we conclude that the blockade of early steps of N-glycosylation during carbohydrate processing resulted in the inhibition of subsequent incorporation of O-linked sugars on CD45 polypeptides, thus preventing the late acquisition of the CD45R0 and CD45RB determinants on the 180- and 190-kDa CD45 polypeptides.

Rapid Communication: Nonspecific Adsorption of Transferrin to K562 Cell Membrane

In the absence of albumin, incubation of K562 cells with radiolabeled transferrin revealed a binding component with high affinity and one with low affinity. The component with low affinity was eliminated in the presence of albumin, indicating it results from non-specific adsorption of ligand to cell membrane. The lack of specificity was further confirmed by demonstration that this component could be dissociated by pronase but not by acid. The high affinity component was responsible for most or all of the iron uptake by the cell. However, in certain cell densities of the culture, some iron could be taken up via other mechanisms. Therefore, the mechanism of low affinity binding could contribute to iron uptake by the cell in certain proliferative states.