We tested the effect of iron deprivation on the uptake of iron from ferric citrate by human erythroleukemia K562 cells. The iron uptake after 24-h preincubation in defined iron-free medium was approximately 2-3x higher than after the preincubation in control transferrin-containing medium. The preincubation of K562 cells in iron-free medium together with the inhibitor of protein synthesis cycloheximide completely abrogated the stimulation of the iron uptake. The preincubation in iron-free medium resulted in a slight decrease (20%) of DMT1 mRNA level. The level of Dcytb, ferroportin and hephaestin mRNA did not exert any significant change. We also did not find any significant effect on the protein level of DMT1, Dcytb, ferroportin and hephaestin. We conclude that iron deprivation stimulates the uptake of non-transferrin iron in K562 cells and that this stimulation depends on protein synthesis. It seems that the expression of an unknown or seemingly unrelated protein(s) is involved.

• MeSH
• buňky K562 MeSH
• časové faktory MeSH
• cykloheximid farmakologie   MeSH
• financování organizované MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• proteiny regulující obsah železa antagonisté a inhibitory   biosyntéza   MeSH
• transferin metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• železo farmakokinetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• srovnávací studie MeSH

Erythropoiesis is a multistep process regulated at the molecular level by intrinsic and extrinsic factors including microRNAs (miRNAs). We previously identified aberrant expression of miR-451 and miR-150 in polycythemia vera (PV) erythroid differentiating cells. To address the functional relevance of these miRNAs in erythroid differentiation, we employed synthetic mimics and inhibitors of miR-451 and miR-150 in erythroid differentiating K562 cells. We observed that miR-451 up-regulation and miR-150 down-regulation are associated with progression of erythroid maturation in K562 cells. Further, enforced expression of miR-451 promoted erythroid differentiation. Inhibition of miR-150 reduced hemoglobinization of K562 cells. Microarray data suggested potential targets regulated by miR-451: UBE2H, ARPP-19; and by miR-150: MS4A3, AGA, PTPRR. Our results demonstrate that miR-451 is involved in the regulation of erythroid differentiation and functions as an enhancer of differentiation. These data support the concept that aberrant expression of miRNAs may contribute to abnormal erythropoiesis such as that of PV.

• MeSH
• akutní erytroblastická leukemie genetika   patologie   MeSH
• buněčná diferenciace účinky léků   genetika   MeSH
• buňky K562 MeSH
• erytroidní buňky účinky léků   patologie   MeSH
• erytropoéza účinky léků   genetika   MeSH
• hemin farmakologie   MeSH
• lidé MeSH
• mikro RNA genetika   fyziologie   MeSH
• nádorové buněčné linie MeSH
• regulace genové exprese u leukemie účinky léků   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• stanovení celkové genové exprese MeSH
• transfekce MeSH
• upregulace účinky léků   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

We have shown previously that iron deprivation significantly stimulates the uptake of non-transferrin ferric iron from ferric citrate by erythroleukemia K562 cells and that this stimulation depends on protein synthesis. However, we have not detected increased expression of any known iron transport protein (Kovar J. et al. (2006) Blood Cells Mol Dis 37:95-99). Therefore, in order to identify membrane proteins of K562 cells with increased expression under iron deprivation, we employed the isolation of membrane proteins by two-phase partitioning system, protein separation by high-resolution 2D electrophoresis, computer differential analysis, and tandem mass spectrometry. Employing these techniques we identified two proteins with statistically significant upregulation, i.e., aldolase A (ALDA) and voltage-dependent anion channel 2 (VDAC2). The upregulation of aldolase A and VDAC2 in K562 cells under iron deprivation was also confirmed by western blot analysis. This is the first time when the control of aldolase A and VDAC2 levels by iron status of the cell is demonstrated.

• MeSH
• aldolasa genetika   metabolismus   MeSH
• buňky K562 MeSH
• deficit železa MeSH
• financování organizované MeSH
• lidé MeSH
• napětím ovládaný aniontový kanál 2 genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• upregulace MeSH
• železo MeSH
• Check Tag
• lidé MeSH

Methyl methanesulphonate (MMS) is a DNA damaging agent, which induces oxidative stress, ATP depletion, and consequently, cell death, in HL-60 and K562 cells. The cell death induced by MMS predominantly exhibited the morphological and biochemical hallmarks of necrosis. A minor population of dying cells exhibited apoptotic hallmarks, especially in K562 cell cultures. Cyclosporin A (CsA) was used to modulate the MMS-induced cell death. Our results indicated that CsA did not prevent cells from dying, but changed the mode of death from necrotic to apoptotic. Surprisingly, CsA enhanced oxidative stress and increased the overall number of dead cells. Based on these results, we conclude that the modulatory effect of CsA on MMS-induced cell death might arise from an interference by CsA with mitochondrial metabolism, rather than from inhibition of the MMS efflux mediated by P-glycoprotein.

• MeSH
• apoptóza účinky záření   MeSH
• buňky K562 enzymologie   patologie   účinky léků   MeSH
• cyklosporin toxicita   MeSH
• DNA nádorová účinky léků   MeSH
• financování organizované MeSH
• glutathion metabolismus   MeSH
• glutathiondisulfid metabolismus   MeSH
• HL-60 buňky enzymologie   patologie   účinky léků   MeSH
• imunosupresiva toxicita   MeSH
• kaspasa 3 biosyntéza   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• methylmethansulfonát toxicita   MeSH
• nekróza chemicky indukované   MeSH
• oxidační stres účinky záření   MeSH
• P-glykoprotein genetika   metabolismus   MeSH
• poškození DNA MeSH
• RNA nádorová analýza   MeSH
• synergismus léků MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH

The present study was undertaken to provide more information on nuclear diameter in leukemic granulocytic early precursors myeloblasts. These cells represented by K562 myeloblasts originated from the blastic phase of the chronic myeloid leukaemia (CML) carry characteristic bcr-abl fusion gene. They represent a convenient model for in vitro studies of CML myeloblasts and are sensitive to various agents which may induce ageing, differentiation and cell death. Mean nuclear diameter (MNuD) and largest nuclear diameter (Mx NuD) in stained cytospins of these cells were measured at a high light microscopic magnification by means of computer programme. Starving cultures were used for induction of ageing without preceding differentiation, sodium butyrate was used as a cytostatic agent or differentiation inducer and imatinib mesylate represented a cytostatic agent for CML. The largest shift of MNuD to smaller values was noted in ageing cultures or cultures treated with butyrate. The decrease of MNuD was less apparent in resistant cells treated with imatinib. This drug, however, produced a very large incidence of necrotic or apoptotic cells or bodies. From the methodical point of view it should be mentioned that values of maximal nuclear diameter (MxNuD) followed similar trends as MNuD and thus provided similar information. The measurement of both nuclear diameters, i.e. MNuD and MxNuD might be a complementary and simple tool to evaluate the cell state in cytological preparations because of their decrease in ageing cells or cells treated with antiproliferative drugs of different mode of action.

• MeSH
• bcr-abl fúzní proteiny genetika   MeSH
• buňky K562 MeSH
• butyráty farmakologie   MeSH
• chronická myeloidní leukemie genetika   MeSH
• financování organizované MeSH
• karyotypizace MeSH
• lidé MeSH
• piperaziny farmakologie   MeSH
• pyrimidiny farmakologie   MeSH
• stárnutí účinky léků   MeSH
• Check Tag
• lidé MeSH

Chronic myeloid leukemia (CML) is a malignant hematopoietic disorder distinguished by the presence of a BCR‑ABL1 fused oncogene with constitutive kinase activity. Targeted CML therapy by specific tyrosine kinase inhibitors (TKIs) leads to a marked improvement in the survival of the patients and their quality of life. However, the development of resistance to TKIs remains a critical issue for a subset of patients. The most common cause of resistance are numerous point mutations in the BCR‑ABL1 gene, followed by less common mutations and multiple mutation-independent mechanisms. Recently, exosomes, which are extracellular vesicles excreted from normal and tumor cells, have been associated with drug resistance and cancer progression. The aim of the present study was to characterize the exosomes released by imatinib‑resistant K562 (K562IR) cells. The K562IR‑derived exosomes were internalized by imatinib‑sensitive K562 cells, which thereby increased their survival in the presence of 2 µM imatinib. The exosomal cargo was subsequently analyzed to identify resistance‑associated markers using a deep label‑free quantification proteomic analysis. There were >3,000 exosomal proteins identified of which, 35 were found to be differentially expressed. From this, a total of 3, namely the membrane proteins, interferon‑induced transmembrane protein 3, CD146 and CD36, were markedly upregulated in the exosomes derived from the K562IR cells, and exhibited surface localization. The upregulation of these proteins was verified in the K562IR exosomes, and also in the K562IR cells. Using flow cytometric analysis, it was possible to further demonstrate the potential of CD146 as a cell surface marker associated with imatinib resistance in K562 cells. Taken together, these results suggested that exosomes and their respective candidate surface proteins could be potential diagnostic markers of TKI drug resistance in CML therapy.

• MeSH
• antigen CD146 metabolismus   MeSH
• antigeny CD36 metabolismus   MeSH
• apoptóza účinky léků   MeSH
• bcr-abl fúzní proteiny antagonisté a inhibitory   genetika   MeSH
• buňky K562 MeSH
• chemorezistence MeSH
• chronická myeloidní leukemie farmakoterapie   genetika   patologie   MeSH
• exozómy účinky léků   metabolismus   MeSH
• imatinib mesylát farmakologie   terapeutické užití   MeSH
• inhibitory proteinkinas farmakologie   terapeutické užití   MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• nádorové buněčné linie MeSH
• proteiny vázající RNA metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), an uncoupler of mitochondrial oxidative phosphorylation, inhibits cell proliferation and induces cell death with apoptotic features. It was reported that the cytotoxic effects of FCCP are preceded by a rapid glutathione (GSH) depletion with a subsequent loss of mitochondrial transmembrane potential (ΔΨ). The GSH depletion was suggested as the cause of apoptosis in FCCP treated cells. This conclusion was further supported by the finding that all adverse effects of FCCP including cell death can be prevented by N-acetylcysteine (NAC) a precursor of GSH synthesis (Han and Park, 2011). Here, we argue that neither loss of ΔΨ nor GSH depletion is sufficient to account for induction of apoptosis in FCCP treated leukemia K562 cells. Indeed, the lowest concentration of FCCP that brings about the permanent loss of ΔΨ and the extensive decrease in GSH level induces cell death in minor population of cells. Only much higher concentrations of FCCP, that exceed the range to achieve permanent collapse of ΔΨ, induce extensive apoptosis. The low proapoptotic activity of FCCP could be explained by hyperactivation of protein kinase B/Akt. A detailed LC/MS/MS analysis of cell extracts revealed extensive formation of FCCP adducts with GSH. This effect could explain the mechanism of GSH depletion, which is currently unknown. Although NAC induces an increase in the GSH pool, this effect is not crucial for abrogation of FCCP cytotoxicity. Indeed, the presence of NAC in the growth medium causes a rapid clearance of FCCP due to its quantitative conversion into the FCCP-NAC adduct, which is the real cause of abrogated FCCP cytotoxicity.

• MeSH
• acetylcystein chemie   farmakologie   MeSH
• apoptóza účinky léků   MeSH
• buňky K562 účinky léků   metabolismus   MeSH
• glutathion chemie   metabolismus   MeSH
• karbonylkyanid-p-trifluormethoxyfenylhydrazon chemie   farmakologie   MeSH
• lidé MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuzelová, D. Grebenová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67-78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT.

• MeSH
• buněčná smrt účinky léků   účinky záření   MeSH
• buňky K562 patologie   účinky léků   účinky záření   MeSH
• časové faktory MeSH
• cytoskelet patologie   účinky léků   účinky záření   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• faloidin chemie   MeSH
• financování organizované MeSH
• fluorescein-5-isothiokyanát MeSH
• fotosenzibilizující látky farmakologie   MeSH
• kyselina aminolevulová farmakologie   MeSH
• lidé MeSH
• Lim-kinasy MeSH
• messenger RNA metabolismus   MeSH
• mikrofilamentové proteiny metabolismus   MeSH
• molekulární chaperony metabolismus   MeSH
• onkogenní proteiny metabolismus   MeSH
• proteinkinasy fyziologie   MeSH
• proteiny tepelného šoku HSP90 metabolismus   MeSH
• regulace genové exprese MeSH
• světlo MeSH
• transkripční faktory MeSH
• transportní proteiny metabolismus   MeSH
• tyrosinkinasy metabolismus   účinky léků   účinky záření   MeSH
• Check Tag
• lidé MeSH

BACKGROUND: Leukaemia is an aggressive cancer of haematopoiesis. Despite increasing treatment success, the relapse rate is still high. Natural killer (NK) cells play a key role in the immune response to malignancies; thus, it is conceivable that NK cell-based immunotherapy may control relapses, while extending the disease-free survival. In our study, we investigated whether cryopreserved NK cells are able to kill the leukaemic K562 cell line, the necessity of IL-2 co-application and the association of activation marker expression (NKp44, NKG2D and CD25) with cytotoxic potential. MATERIALS AND METHODS: K562 cells were added to NK cell cultures in different ratios, i.e. 1:5, 1:10 and 1:20 (K562/NK), immediately after thawing NK cells or after 3-6-12-24 h of re-cultivation with or without IL-2. RESULTS: Our results demonstrated the ability of cryopreserved NK cells to kill K562 in all ratios, times and culture conditions. The number of dead K562 cells depended on the number of NK cells and on the presence of IL-2. NK cells cytotoxic potential decreased gradually in the culture without IL-2. In contrast, NK cell-mediated cytotoxicity remained the same during the entire re-culture period after IL-2 re-application. CONCLUSION: Our study proved the efficacy of using cryopreserved ready-for-use NK cells in relapse treatment and the need for simultaneous administration of IL-2.

• MeSH
• aktivace lymfocytů MeSH
• buňky K562 MeSH
• buňky NK imunologie   MeSH
• cytotoxicita imunologická * MeSH
• imunoterapie adoptivní metody   MeSH
• kryoprezervace * MeSH
• kultivované buňky MeSH
• leukemie terapie   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The effect of P-glycoprotein (P-gp, ABCB1, MDR1) expression on cell resistance to nilotinib was studied in human leukaemia cells. We used K562/Dox cells overexpressing P-gp and their variants (subclones) with a gradually decreased P-gp expression. These subclones were established by stable transfection of K562/Dox cells with a plasmid vector expressing shRNA targeting the ABCB1 gene. Functional analysis of P-gp using a specific fluorescent probe indicated gradually decreased dye efflux which was proportional to the P-gp expression. We observed that K562/Dox cells overexpressing P-gp contained a significantly reduced intracellular level of nilotinib when compared to their counter partner K562 cells, which do not express P-gp. This effect was accompanied by a decreased sensitivity of the K562/Dox cells to nilotinib. Importantly, cells with downregulated expression of P-gp gradually lost their ability to decrease the intracellular level of nilotinib although they still significantly decreased the intracellular level of daunorubicin (DNR). Accordingly, cells with the reduced expression of P-gp concomitantly failed to provide resistance to nilotinib, however, they exhibited a significant resistance to DNR. Taken together, we demonstrated that the conclusion as to whether P-gp is involved in nilotinib resistance or not strongly depends on its expression at protein level.

• MeSH
• buňky K562 MeSH
• chemorezistence účinky léků   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• leukemie farmakoterapie   metabolismus   MeSH
• lidé MeSH
• P-glykoprotein metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky farmakologie   MeSH
• pyrimidiny farmakologie   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH