Treatment Of K562 Cells Research Articles

Extracts of Medicinal Mushrooms Agaricus bisporus and Phellinus linteus Induce Proapoptotic Effects in the Human Leukemia Cell Line K562

We have studied the effects of Agaricus bisporus and Phellinus linteus ethanol extracts on transcriptional regulation of genes involved in cytokine release and apoptosis in the human leukemia cell line K562. In particular, we applied quantitative real-time PCR (Q-PCR) assays to monitor altera- tions of gene expression for the following genes: Bcl-2, Casp-9, NF-κB, TNF-α, IFN-γ, and IL-10. We found stronger proapoptotic activity for the Ph. linteus alcohol extract on K562 cells than for the A. bisporus extract: 4.4- and 2.2-fold increase of Bcl-2 and Casp-9 transcripts. Mushroom alcohol ex- tracts are suggested to exert their effects on tumor cells via the induction of apoptosis. K562 leukemia cells were shown to be most responsive to the transcriptional induction of tumor necrosis factor TNF-α when stimulated with IFN-γ and then treated with Ph. linteus alcohol extract: up to a 4.5-fold increase. Treatment of K562 cells with A. bisporus extract promoted transcription of the cytokine gene IL-10.

Implication of unfolded protein response in resveratrol-induced inhibition of K562 cell proliferation

Resveratrol (RES), a natural plant polyphenol, is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, RES has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventive and anti-tumor agent in vivo. The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, the mechanisms by which RES mediates its effects are not yet fully understood. In this study, we showed that RES caused cell cycle arrest and proliferation inhibition via induction of unfolded protein response (UPR) in human leukemia K562 cell line. Treatment of K562 cells with RES induced a number of signature UPR markers, including transcriptional induction of GRP78 and CHOP, phosphorylation of eukaryotic initiation factor 2α (eIF2α), ER stress-specific XBP-1 splicing, suggesting the induction of UPR by RES. RES inhibited proliferation of K562 in a concentration-dependent manner. Flow cytometric analyses revealed that K562 cells were arrested in G1 phase upon RES treatment. Salubrinal, an eIF2α inhibitor, or overexpression of dominant negative mutants of PERK or eIF2α, effectively restored RES-induced cell cycle arrest, underscoring the important role of PERK/eIF2α branch of UPR in RES-induced inhibition of cell proliferation.

Induction of γ-Globin Gene Expression Via the Nrf2/Antioxidant Response Element Signaling Pathway.

Abstract 975Induction of fetal hemoglobin (HbF) has proven therapeutic potential to treat sickle cell disease and β-thalassemia. However, agents known to be effective in humans, including HU, DNMT inhibitors and butyrate derivatives are not ideal due to suppression of hematopoeisis and the possibility of long-term side effects including those related to DNA mutation and epigenetic changes. Recently, two natural compounds, angelicin (Lampronti, et al, Eur J Hematol 2003) and resveratrol (Rodrigue, et al, Hematology 2001) have been found to induce γ-globin gene expression in K562 cells. These agents may be important lead compounds as they are generally non-cytotoxic and are being evaluated in ongoing human trials as cancer chemoprevention agents. These and several other agents are thought to work by activating antioxidant response pathway genes. The products of these genes are enzymes involved in antioxidant and detoxification activities and include NADPH-quinone oxireductase 1 (NQO1), glutamate-cysteine ligase (GCL) and glutathione S-transferase (GST). The activation of these genes is mediated by the transcription factor NF-E2 related factor 2 (Nrf2) which binds to a specific antioxidant response element (ARE) sequence (TGACnnnGCA) in target gene promoters. The proximal γ-globin promoter contains an ARE sequence between two CAAT boxes, suggesting that it too may be activated by Nrf2. This led us to hypothesize that drugs which activate the ARE/Nrf2 pathway may provide a less toxic approach to HbF induction. Several compounds that activate this pathway are already approved for human use or are under investigation in human trials. To test this hypothesis, we treated K562 cells with various Nrf2 pathway activators. First, we determined the maximum concentration of each compound that did not inhibit proliferation of K562 cells. Using these doses, we performed time course experiments by treating K562 cells and then measuring steady-state mRNA levels of Nrf2 target genes and γ-globin using quantitative real-time PCR. We initially tested six compounds that are known to induce antioxidant response genes. We found the most pronounced γ-globin induction followed treatment with tert-butylhydroquinone (tBHQ) (2.8 fold), 3H-1,2 dithiole-3-thione (D3T) (2.4 fold) and curcumin (2.1 fold). We next tested tBHQ in erythroid precursors isolated from normal human bone marrow. In these primary cells it also significantly increased expression of γ-globin mRNA and of Nrf2 target genes NQO1, GCLM and GSTP1. To determine if Nrf2 was necessary for tBHQ induction of γ-globin mRNA we used siRNA to knockdown Nrf2 expression in K562 cells. Our results show that transiently silencing Nrf2 transcription prevented γ-globin and NQO1 gene induction by tBHQ compared to samples transfected with scrambled siRNA (p < 0.01). To determine if this requirement for Nrf2 is due to Nrf2 binding at the ARE consensus sequence in the γ-globin promoter, ChIP analysis was performed. This demonstrated that Nrf2 binding at both the γ-globin and NQO1 promoters was progressively increased at 4 and 6 hours after tBHQ treatment compared to the negative control necdin promoter where there was no change. Taken together, these results suggest that the antioxidant tBHQ induces γ-globin mRNA expression through the Nrf2/ARE pathway and that this may be a less toxic strategy for γ-globin gene induction in people with hemoglobinopathies. Disclosures:No relevant conflicts of interest to declare.

Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.

Abstract 4250Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5×10 7 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration). Gene expression data of the treated cells were compared with data of untreated cells. In addition, proliferation (MTS Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. Protein levels of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2 were detected with Western blots. Particular emphasis was put on 303 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in vivo during treatment with Imatinib (Bruennert et al, Leukemia, 2008). In K562 cells, we found that Imatinib led to a significant differential expression of 45 of those genes. In general, the effect of Nilotinib with regard to the number of genes affected and degree of suppression was more pronounced resulting in a significant differential expression of 120 genes of the aforementioned genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib. Downregulation of genes involved in cell cycle was observed in 17 genes following Nilotinib exposure, but only in the PIM-1 gene following Imatinib exposure. This effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5-fold greater following treatment with Nilotinib in comparison to treatment with DMSO, whereas Imatinib treated K562 cells had a 1.3-fold higher apoptosis rate compared to DMSO treated 562 cells after 24 hours. The superiority of Nilotinib is also reflected by the results of protein analysis of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2: Both tyrosine kinase inhibitors completely inhibit the phosphorylation of STAT5, but Nilotinib inhibits PIM-1 and PIM-2 stronger than Imatinib (by 85% vs. 70%) in K562 cells. In summary, on a molecular level Nilotinib is apparently more potent than Imatinib with regard to the number of genes affected and the degree of their suppression. Among the 45 genes that were significant differentially expressed with both drugs, genes of Imatinib treated K562 cells were downregulated 1.68fold (mean) whereas genes of Nilotinib treated K562 cells were downregulated 2.41fold (mean). The genes altered are mainly associated with cell cycle regulation. In addition to this Nilotinib has a stronger inhibitory effect on PIM 1 and 2. Disclosures:Gattermann:Novartis: Honoraria, Participation in Advisory Boards on deferasirox clinical trials.

Rab7b Promotes Megakaryocytic Differentiation of K562 Cells by Activating the Protein Kinase C/Extracellular Signal-Regulated Kinase Dependent Pathway.

Abstract 3612Poster Board III-548Rab proteins are small GTPases belonging to the Ras superfamily and involved in cell protein transportation processes such as internalization and exocytosis. Recently it has been found that several Rab proteins such as Rab7 are also essential for signaling and the controlling of cell proliferation and differentiation. We previously have identified a small GTPase homologous to Rab7, named Rab7b, which is localized to lysosome-associated compartments and is selectively expressed in promyeloid or monocytic cells. We demonstrated that rab7b is involved in monocytic differentiation of human acute promyelocytic leukemia cells. In this study, we detected the expression level of Rab7b during the process of PMA induced megakaryocytic differentiation of K562 cells, transfected Rab7b and its mutants into K562 cells, and investigated the function and signaling mechanism of PMA induced megakaryocytic differentiation of K562 cells. Firstly, we treated K562 cells with PMA for various times, detected the expression level of Rab7b by quantitative PCR and Western blot, and measured the expression level of CD11b, CD14, CD33 and CD41a on the cell surface. Next, we constructed Rab7b, Rab7b-Q67L (active GTPase-deficient mutant), Rab7b-ΔCC (localization deficiency) and Rab7b-RNAi expression vectors, which were then transfected into K562 cells. Megakaryocytic differentiation of cells were analyzed after treated with PMA for various times, including cell morphology and adhesive properties, cell growth arrest, polyploidization, secretion of IL-6 and expression of specific marker CD41a. Because of the considerable role of PKC and ERK1/2 signaling pathways in the megakaryocytic differentiation of K562 cells, we treated various K562 cells with PMA for various times and detected the activation of ERK1/2 by Western blot. Later, cells were inhibited by PKC and ERK1/2 specific inhibitors and treated with PMA for various times, and then the expression of Rab7b and megakaryocytic differentiation of K562 cells were observed. At last, PMA-induced activation of PKC and ERK1/2 signaling has been suggested to regulate cell cycle arrest and megakaryocyte differentiation by modulating the expression of the p21waf/cip1 and the dephosphorylation of the Rb protein, which were then detected by us after transfection of Rab7b and inhibition of PKC/MAPK pathways in K562 cells. The expression of Rab7b was upregulated during the PMA-induced megakaryocytic differentiation of K562 cells. Overexpression of wild-type and active mutant (Q67L) of Rab7b enhanced PMA stimulated megakaryocytic differentiation and ERK1/2 activation. In addition, overexpression of the localization deficient mutant (ΔCC) or RNAi of Rab7b inhibited PMA-stimulated megakaryocytic differentiation and ERK1/2 activation. PMA-induced megakaryocytic differentiation and activation of ERK1/2 in K562 cells was reversed by inhibiting either PKC or ERK1/2 signaling pathway, whereas the mRNA and protein expression of Rab7b was only suppressed in PKC inhibiting groups, indicating that Rab7b mediates a signal for megakaryocytic differentiation downstream of PKC and upstream of ERK1/2. Therefore, we concluded that Rab7b could promote PMA induced differentiation of K562 cells by activating the PKC/ERK1/2 signaling pathway and provide Rab7b as a candidate to connect the PKC pathway and the ERK1/2 pathway in PMA-induced megakaryocytic differentiation of K562 cells. Disclosures:No relevant conflicts of interest to declare.

Deranged MicroRNA 16-2 Expression Contributes to Erythropoiesis in Polycythemia Vera.

Abstract 3896Poster Board III-832We previously reported the finding of significantly raised levels of mature microRNA 16 (miR-16) in CD34+ cells from PV patients (pts) (Guglielmelli P et al, ASH 2008, 179A). Mature miR-16 can derive from both miR-16-1 at chr13q14.3 and miR-16-2 at chr3q26.1, which differ in their precursor miRNA (pre-miR) sequence. To distinguish between either genes as a source of increased miR-16 levels we quantified each pre-miR in PV CD34+; we observed that the premiR-16-1/16-2 ratio decreased from 1.47± 2.38 in controls (ctr) to 0.52± 0.34 in PV (P<.001). To confirm the preferential expression of miR-16-2 we knocked-down miR-16-1 or miR-16-2 using specific siRNAs; in PV CD34+ treated with miR16-2 siRNA the miR-16 levels were 90±9% lower than in scramble-treated cells while miR16-1 siRNA caused only a 10±3% reduction (P <.01). Levels of miR-16 in PV CD34+ were not correlated to the JAK2V671F burden; also, there was no modification of miR-16 in JAK2V617F mutant HEL or UKE-1 cells after treatment with the JAK2 inhibitor AZD1480. These data suggested that the abnormally increased mature miR-16 in PV are largely derived from miR-16-2. To address underlying mechanisms we performed direct sequencing at the miR-16-1 and miR-16-2 regions without detecting sequence abnormalities; also we excluded gene copy number changes (SNP 6.0 array). The promoter of miR16-2 is not characterized yet, but recent data suggest that it might be the same of the structural maintenance of chromosomes-4 (SMC4) gene, to which miR16-2 is intronic. We have found a significant correlation between miR-16 and SMC4 mRNA levels in PV CD34+ (R=0.77, P<.001); furthermore, after induced erythroid differentiation of normal CD34+ a concurrent increase of miR-16 and SMC4 mRNA was observed, indirectly supporting a shared regulatory control. Methylation-specific PCR and sequencing at upstream CpG-rich regions discovered that PV patients had unmethylated CpGs compared to control cells, suggesting that methylation may concur to miR-16-2 regulated expression. To evaluate involvement of miR16 in abnormal erythropoiesis of PV, we first analyzed the kinetics of miR-16 during erythroid differentiation of CD34+. Levels of miR-16 steadily increased from day 6, when progenitors were switched from proliferative to differentiative phase, up to day 12-14 and, at any time point considered, those measured in PV cells were significantly greater than in ctrs (P= .01). Also, a time-dependent increase of miR16 paralleling the transcription of beta-globin mRNA was observed in hydroxyurea or Na-butyrate treated K562 cells. Then we over-expressed (Amaxa) mature miR16 in normal CD34+ at day 6 of culture, and found that the percentage of CD36+/GPA+ cells increased from 13.6±4.8% in scramble to 28.8±11.3% (P<.05); notably, even in the absence of EPO, some GPA+ cells were generated from miR-16-transduced CD34+. Furthermore, normal CD34+ cells transfected with miR-16 produced significantly increased number of CFU-e and BFU-E (174±100 and 1283±250/103 CD34+) compared to scramble cells (105±90 and 733±90; P<.01) while myeloid colonies were unchanged. A genome-wide expression profiling in normal CD34+ transfected with miR-16-2 siRNA discovered 618 genes significantly de-regulated, among which genes involved in erythroid differentiation such as NFE2, MYB, FGFR2, HLF, GATA-1 and globin family genes (HBA1, HBA2, HBB). Overall, these data support a role of miR-16 in normal erythropoiesis. In case of PV we employed a knock-down strategy using either pre-miR-16-1 or pre-miR-16-2 siRNAs. The use of miR-16-2 siRNA resulted in significant reduction of Epo-independent erythroid colonies (EEC) from GFP-sorted CD34+ cells (60±18% lower than scramble) while no change was observed in cell treated with anti-miR-16-1. These data were confirmed in independent experiments using peripheral blood mononuclear cells (MNC): the number of EEC decreased from 207±20 to 170±23 to 95±25/105 MNC in cells transduced with scramble, anti miR-16-1 or miR-16-2, respectively (P<.01 for miR-16-2 vs others). Also the number of BFU-E generated in cytokine-supplemented cultures of PV MNC was significantly reduced by miR-16-2 siRNA (360±80) compared to cells transduced with scramble (540±120) or miR-16-1 (500±87) (P<.001). In summary, these data support a role for deregulated miR-16-2 in abnormal erythropoiesis of PV, and anticipate its possible relevance as a therapeutic target. Disclosures:No relevant conflicts of interest to declare.

A non‐apoptotic function of caspase‐3 in pharmacologically‐induced differentiation of K562 cells

Several anticancer drugs with diverse chemical structures can induce differentiation of cancer cells. This study was undertaken to explore the potential contribution of caspase-3 to pharmacologically-induced differentiation of K562 cells. We assessed differentiation by measuring the expression of glycophorin A and haemoglobin synthesis in K562 cells treated with low concentrations of doxorubicin, hydroxyurea, cytosine arabinoside, cisplatin and haemin. Caspase-3 activation, mitochondrial membrane potential dissipation and viability were assessed by FACS. GATA-1-binding activity was evaluated by EMSA. Treatment of K562 cells with low concentrations of the tested drugs activated caspase-3 but did not trigger detectable apoptosis. Instead, elevated levels of haemoglobin-positive and glycophorin A/caspase-3-double-positive cells were observed, suggesting involvement of caspase-3 in drug-induced differentiation. Inhibition of caspase-3 activity significantly reduced the ability of K562 cells to execute the differentiation programme. Mitochondrial membrane potential dissipation was observed, indicating involvement of the mitochondrial pathway. Binding activity of GATA-1, transcription factor responsible for differentiation and cell survival, was not diminished by increased caspase-3 activity during drug-stimulated differentiation. Our results could explain how anticancer drugs, with diverse structures and modes of action, can stimulate erythroid differentiation in leukaemic cells with appropriate genetic backgrounds. Our findings imply that some similarities exist between pharmacologically-induced differentiation of erythroleukaemic cells and normal erythropoiesis, both involving caspase-3 activation at high levels of anti-apoptotic protein Bcl-X(L) and chaperone protein Hsp70 (heat shock protein 70). Therefore, the functions of caspase-3, unrelated to cell death, can be extended to pharmacologically-induced differentiation of some cancer cells.

Treatment of K562 cells with 1,25-dihydroxyvitamin D3 induces distinct alterations in the expression of apoptosis-related genes BCL2, BAX, BCLXL, and p21

Apoptosis, or programmed cell death, is a very important phenomenon in cytotoxicity induced by anticancer treatment. 1α,25-Dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), the active metabolite of vitamin D, inhibits the growth of multiple types of cancer cells including breast, colon, and prostate cancer cell lines. We studied alterations in the mRNA expression levels of BCL2, BAX, CYC, BCL-XL, and VDR genes in the K562 chronic myeloid leukemia cell line in response to treatment with 1,25-(OH)(2)D(3). Morphological observation of K562 cells was evaluated by the staining with Wright's solution. Cell percentage at different phases of the cell cycle was measured, and apoptosis was measured by flow cytometry. The expression levels of the apoptosis-related genes were analyzed by real-time reverse transcription polymerase chain reaction. We found that treatment with 1,25-(OH)(2)D(3) down-regulates BCL2 and BCL-XL mRNA expressions, as well as up-regulates expressions of BAX and p21 mRNA. The expression pattern of CYC and VDR genes were not influenced. However, K562 cells treated with 1,25-(OH)(2)D(3) caused an arrest of cell cycle progression in G1 phase resulting in a decreased number of cells in the S phase, complemented by an accumulation of cells in the G0-G1 phases. Our data show the modulatory effects of 1,25-(OH)(2)D(3) treatment in apoptosis-related genes in K562 cells.

Regulation of megakaryocytic differentiation of K562 cells by FosB, a member of the Fos family of AP-1 transcription factors.

The regulation of megakaryocytic differentiation is poorly understood. Using K562 cells, which can partly recapitulate the process in response to phorbol 12-myristate 13-acetate (PMA), we performed microarray-based gene expression profiling to identify genes that play significant roles in megakaryopoiesis. Here, we describe the function of FosB, an AP-1 transcription factor. FosB is induced in PMA treated K562 cells in a sustained manner and forms an active AP-1 protein-DNA complex. Down-regulation of FosB with specific shRNAs inhibited the induction of CD41, a specific cell surface marker of megakaryocytes. We also show that activation of the PKC-MEK-ERK signaling pathway is required for induction of FosB and CD41. Finally, we cross-examined the microarray data in conjunction with gene function annotation data to identify additional target genes of FosB. We define 3 genes, INHBA, CD9, and ITGA2B as regulatory targets of FosB and show that CD9, in particular, is a direct target of FosB.

Induction of endogenous γ-globin gene expression with decoy oligonucleotide targeting Oct-1 transcription factor consensus sequence

Human β-globin disorders are relatively common genetic diseases cause by mutations in the β-globin gene. Increasing the expression of the γ-globin gene has great benefits in reducing complications associated with these diseases. The Oct-1 transcription factor is involved in the transcriptional regulation of the γ-globin gene. The human γ-globin genes (both Aγ and Gγ-globin genes) carry three Oct-1 transcription factor consensus sequences within their promoter regions. We have studied the possibility of inducing γ-globin gene expression using decoy oligonucleotides that target the Oct-1 transcription factor consensus sequence. A double-stranded 22 bp decoy oligonucleotide containing the Oct-1 consensus sequence was synthesized. The results obtained from our in vitro binding assay revealed a strong competitive binding of the decoy oligonucleotide for the Oct-1 transcription factor. When K562 human erythroleukemia cells were treated with the Oct-1 decoy oligonucleotide, significant increases in the level of the γ-globin mRNA were observed. The results of our western blots further demonstrated significant increases of the fetal hemoglobin (HbF, α2γ2) in the Oct-1 decoy oligonucleotide-treated K562 cells. The results of our immunoprecipitation (IP) studies revealed that the treatment of K562 cells with the Oct-1 decoy oligonucleotide significantly reduced the level of the endogenous γ-globin gene promoter region DNA co-precipitated with the Oct-1 transcription factor. These results suggest that the decoy oligonucleotide designed for the Oct-1 transcription factor consensus sequence could induce expression of the endogenous γ-globin gene through competitive binding of the Oct-1 transcription factor, resulting in activation of the γ-globin genes. Therefore, disrupting the bindings of the Oct-1 transcriptional factors with the decoy oligonucleotide provides a novel approach for inducing expression of the γ-globin genes. It also provides an innovative strategy for the treatment of many disease conditions, including sickle cell anemia and β-thalassemia.

Methoxy VO-salen complex: In vitro antioxidant activity, cytotoxicity evaluation and protective effect on CCl 4-induced oxidative stress in rats

Reactive oxygen species (ROS) play crucial roles in normal physiological processes. However, the overproduction of ROS is involved in the onset of many degenerative diseases. Regarding this fact, discovery of new antioxidants is interesting for many research groups. In this study, we evaluated the antioxidant properties of a methoxy VO-salen (MetVO-salen) complex employing various in vitro systems. In addition, the cytotoxic effect of MetVO-salen was assessed based on MTT in treated K562 cells. In an in vivo approach, the protective effect of MetVO-salen against CCl 4-induced oxidative stress in rats was also investigated in terms of superoxide dismutase (SOD) and catalase (CAT) activities, as well as in terms of the levels of malondialdehyde (MDA) and glutathione (GSH). Our results indicated that MetVO-salen has an effective capability in scavenging superoxide ( O 2 − ) and hydrogen peroxide (H 2O 2) radicals in a dose-dependent manner. In vivo results also showed that the administration of MetVO-salen to the CCl 4-treated rats caused a significant (222%) increase in SOD activity, a 59% enhancement in GSH content and a 31% decrease in the level of MDA compared to the CCl 4-treated control rats. Overall, MetVO-salen appears to be an effective antioxidant and is quite suitable for further biological evaluation.

PTEN and rapamycin inhibiting the growth of K562 cells through regulating mTOR signaling pathway

ObjectiveTo investigate, in vitro, the regulatory effects of tumor-suppressing gene PTEN on mTOR (mammalian target of rapamycin) signaling pathway, the effects of transfected PTEN and rapamycin on the growth inhibition, and apoptosis induction for human leukemia cell line K562 cells.MethodsK562 cells were transfected with recombined adenovirus-PTEN vector containing green fluorescent protein (Ad-PTEN-GFP), followed by the treatment of the cells with or without rapamycin. The proliferation inhibition rate and apoptotic rate of these transfected and/or rapamycin treated K562 cells were measured by MTT assay and flow cytometry (FCM), the expression levels of PTEN-, mTOR-, cyclinD1- and P27kip1- mRNA were measured by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR), the protein expression levels of PTEN, Akt, p-Akt were detected by western blotting.ResultsThe proliferation of K562 cells was inhibited by PTEN gene transfection with/without the treatment of rapamycin. The expression levels of PTEN- and P27kip1- mRNA were up-regulated, and the mTOR- and cyclinD1- mRNA were down-regulated in K562 cells after the cells transfected with wild type PTEN gene and treated with rapamycin.ConclusionPTEN and rapamycin inhibited mTOR expression by acting as an upstream regulator of mTOR. Low dose rapamycin in combination with over-expressed PTEN might have synergistic effects on inhibiting the proliferation and promoting apoptosis of K562 cells.

Growth inhibitory and differentiation effects of chloroquine and its analogue on human leukemic cells potentiate fetal hemoglobin production by targeting the polyamine pathway

Elevated arginase activity has been implicated in several pathological conditions in sickle cell disease (SCD) and other inflammatory disorders. Recently, we showed that chloroquine (CQ), an anti-malarial and anti-rheumatoid drug, displays a competitive mode of inhibition on sickle erythrocyte arginase. However, the effects of CQ and its analogue, hydroxychloroquine (HCQ) on erythroid differentiation leading to induced fetal hemoglobin (Hb F) production is unknown. In the present study, we obtained evidence of the anti-proliferative and differentiation effects of CQ and HCQ at pharmacologically attainable concentrations. This differentiation effect was linked to a dose-dependent inhibition of arginase activity and induced hemoglobinization, as Hb F synthesis was increased by 3.4- and 3.2-fold for CQ or HCQ, respectively. Treatment of K562 cells with lipopolysaccharide (LPS) or 8-bromo-cAMP (Br-cAMP) failed to reverse the inhibitory effects of CQ or HCQ on arginase activity. Indeed, the combination of Br-cAMP with CQ in LPS-treated cells resulted in a significant enhancement of Hb F and total hemoglobin production. Further, we showed that CQ or HCQ maximally stimulated intracellular cGMP levels by 6.6- and 3.0-fold at 6 and 3 h, respectively, as demonstrated by immunosorbent assay. However, co-treatment of K562 cells with CQ or HCQ in the presence of inhibitors of sGC-PKG-pathways reduced Hb F stimulation, suggesting the possible involvement of the sGC-PKG pathway. This is the first evidence demonstrating the capacity of anti-rheumatoid drugs to modulate the arginine-pathway and result in the enhancement of Hb F production, and thus may provide a paradigm for targeted therapy of hemoglobinopathies and other inflammation-related disorders.

Characterization of Histone Deacetyalses Involved in γ-Globin Gene Regulation.

Fetal hemoglobin (HbF; α2γ2) ameliorates vaso-occlusive symptoms in individuals with sickle cell disease (SCD) because of its ability to inhibit hemoglobin S polymerization. One mechanism for γ-globin reactivation likely involves chromatin modification and the release of repressor complexes in which histone deacetylases (HDACs) may be present. The objective of this study was to identify HDACs involved in γ-globin gene regulation. Experiments were performed in K562 cells to determine the ability of NaB (2mM), TSA (0.5μm) and the non-HDAC-inhibitor γ-globin activator hemin (50μM), to alter transcription levels of the HDAC genes during concomitant HbF induction. Gene expression was measured by reverse transcription (RT) of mRNA followed by quantitative PCR (qPCR) analysis using gene-specific primers. Treatment of K562 cells with TSA and NaB reduced transcription levels of both HDAC9 and HDRP (histone deacetylase related protein, a splice variant of HDAC9) from 20–80% as did the control agent hemin (p<0.05). By contrast, expression of HDAC7 and HDAC10 was enhanced in the presence of both HDAC inhibitors. The altered HDAC gene expression levels provided indirect evidence for a possible role in mechanisms of γ-globin response to drug inducers. Subsequent experiments were performed to delineate whether HDAC9 and HDRP are directly involved in γ-globin regulation. We performed siRNA knockdown of HDAC9 and HDRP in K562 cells to determine the effect on expression of endogenous γ-globin. siRNA oligonucleotides were transfected using Oligofectamine (Invitrogen) for 48 hrs and expression of targeted genes were quantified by RT-qPCR. siHDAC9 and siHDRP (Dharmacon) treatment resulted in dose-dependent γ-globin silencing and transactivation respectively at 80–320nM. Experiments were then performed with 160nM of siHDAC9 or siHDRP in the K562 cell lines which were stably transfected with a luciferase reporter (pGL4.17-Luc2-neo, Promega) under the control of Gγ-globin promoter (−1500 to +36) and the pGL4.17-Luc2-neo empty vector. We likewise observed a 30% decrease in luciferase activity with siHDAC9 and a 40% increase with siHDRP suggesting that HDAC9 and its variant are directly involved in regulation of γ-promoter activity. In summary, the effects on endogenous γ globin levels and Gγ globin promoter activity through HDAC9/HDRPspecific knockdown by siRNA experiments suggest that HDAC9 molecules play a role in regulating γ-globin gene expression. We conclude that HDAC9 and HDRP have opposite regulatory effect on γ-globin gene expression and may act by a feedback mechanism.

Apoptosis Was Induced by Casticin in Acute Myelocytic Leukemia Cells

Casticin, a component from Vitex rotundifolia wich was widely used as an anti-inflammatory agent in Chinese traditional medicine, was reported to have anti-tumor activities in lung cancer and breast cancer. There are yet no reports on roles against acute myelocytic leukemia (AML). This study aims to elucidate the anti-leukemic activity of casticin on AML cells. We investigated the efficient efficacy and the mechanisms by which casticin triggers cell death in AML cells by analyzing cell cycle perturbations, apoptosis-related marker expression. Cell viability was measured by MTT method; apoptosis and cell cycle arrest were determined by flow cytometry and AV-PI assay. Western blot was performed to measure the apoptosis-related marker. Concentration-dependant cell deaths were observed in AML cell lines including K562, U937 and THP-1, with IC50 values of 24h (hours) being 47.4μM, 67.8μM and 61.7μM, respectively. Time-dependant cell deaths were also observed. At the concentration of 20μM casticin, 45.7%, 76.1% and 80.9% of K562 cells were inhibited at 24h, 48h and 72h, respectively; 24.7%, 30% and 61% of U937 cells were inhibited at 24h, 48h and 72h, respectively; while for THP-1, 29%, 41.8% and 53.9% were inhibited at 24h, 48h and 72h, respectively. Apoptosis was found using AV-PI staining by flow cytometry analysis. We observed an obvious G2/M phase increase prolongation in casticin treated K562 cells. BThe distribitions of G2/M phase were 2.9%, 33.6%, 75.3%, 54.9%, 29.7% and 27.0% in K562 cells after treated by 20μM casticin for 0h, 6h, 12h, 24h, 36h and 48h, respectively. Furthermore, apoptosis-related proteins, PARP and caspase 3, were cleaved in casticin treated K562 cells. Taken together, these results demonstrated that casticin can induce leukemic cell death through apoptosis, suggesting that casticin could be a promising therapeutic agent against acute myeloid leukemia.

Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia (CML) cells

This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/Abl-positive chronic myelogenous leukemia (CML) cells (e.g., K562, LAMA84). Treatment of K562 cells with shikonin (e.g., 0.5 muM) resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species (ROS), striking activation of c-Jun-N-terminal kinase (JNK) and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events (i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis) following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.

Growth inhibition and apoptosis of human leukemia K562 cells induced by Seleno-short-chain chitosan

In this article, the in vitro effects of seleno-short-chain chitosan (SSCC, molecular weight between 5,000 and 10,000 Da) on the proliferation of human leukemia K562 cells were investigated to illustrate the possible mechanisms involved. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays showed that short-chain chitosan significantly suppressed the growth of K562 cells in a dose-dependent and time-dependent manner, although normal mouse embryonic fibroblasts NIH3T3 were viable after the same treatment. Cell growth inhibitory rate could reach 95% after exposure with more than 100 microg/ml SSCC for 72 h. Flow cytometry analysis revealed that treatment of K562 cells with SSCC resulted in the accumulation of cells in the G(2)/M phase. The growth inhibition of K562 cells after treatment with SSCC was subsequently associated with the induction of apoptosis, as evidenced by (1) the typical apoptotic morphologic changes measured by a fluorescence microscope, (2) the internucleosomal DNA fragmentation into a ladder determined by agarose gel electrophoresis and (3) the occurrence of sub-G(0)/G(1) phase cells analyzed by flow cytometry. All these results indicated that SSCC may have therapeutic potential in human leukemia treatment.

Effective Telomerase Inhibition Via Expression of Dominant-Negative HTERT Leads to Altered Growth Kinetics, Elevated Apoptosis and Increased Radiosensitivity of Bcr-Abl-Positive K562 Cells In Vitro.

Disease progression in CML is associated with accelerated telomere shortening caused by increased turnover of Bcr-Abl positive cells. We have recently proposed a model in which continuous telomere shortening is correlated with increasing genetic instability in Bcr-Abl-positive cells thus facilitating the acquisition of secondary genetic abnormalities and, as a consequence clonal succession and eventually clinical progression of the disease. Based on this hypothesis, telomerase upregulation in late stage disease is a prerequisite for prevention of replication-induced senescence in Bcr-Abl positive cells. As a consequence, treatment of CML with Telomerase inhibitors (TI) represents an attractive strategy aiming at the potential eradication of cycling CML stem cells. Therefore, we have exploited both pharmacological (small molecule inhibitor) and genetic strategies (dominant negative hTERT mutants, DN-hTERT) of telomerase inhibition in CML cells in vitro We first treated K562 cells with the pharmacological telomerase inhibitor BIBR1532 in vitro. After around 400 population doublings (PD), no differences in growth kinetics nor signs of senescence or apoptosis were observed in BIBR1532 treated cells despite of significant telomere shortening (22 base pairs (bp) per PD) compared to control cells. Furthermore, neither significant differences in mRNA expression of telomere/telomerase-associated proteins, nor accumulation of double strand breaks (DSBs) under irradiation was observed in treated cells with short telomeres as opposed to untreated control cells. The very slow shortening rate of 22bp/PD plus the lack of stigmata pointing to induction of senescence in K562 cells lead to the assumption that telomerase activity is not complete inhibited by the compound. In order to verify the potency of telomerase directed treatments in CML, we therefore expressed DN-hTERT in K562 cells. Integration of DN-hTERT led to a significant decrease in telomerase activity (measured by RQ-TRAP). Furthermore, DN-hTERT expressing cells underwent accelerated telomere shortening at a substantially higher rate (>100 bp/PD) from 15kb to around 4kb within 110 days of culture. In contrast to BIBR1532-treated cells, DN-hTERT expressing K562 cells slowed down growth kinetic in comparison to control cells after 80 days of culture. By using Annexin 5 staining, 25% of apoptotic cells could be detected in cells with critically short telomeres as compared to control cells (<3%). Finally, a significantly increased accumulation of double strand breaks (DSBs) detected by gammaH2AX foci after exposure to irradiation was observed in DN-hTERT K562 cells as compared to control cells pointing to an impaired DNA repair machinery in Bcr-Abl positive cells with disrupted telomere maintenance. In summary, the data suggest that pharmacological telomerase inhibition by BIBR1532 is insufficient to induce telomere-mediated senescence in Bcr-Abl-positive cells. However, accelerated telomere shortening, slowing down of growth kinetics, elevated apoptosis and increased radiosensitivity induced by expression of DN-hTERT indicate a therapeutic potential for telomerase-directed treatment strategies in CML.

Reprogramming of nuclear proteasomes under apoptosis induction in K562 cells I. Effect of glutathione-depleting agent diethylmaleate

In the present work, changes in the subunit composition, phosphorylation state, and enzymatic activities of 26S proteasomes undergoing programmed cell death were studied. Apoptosis in proerythroleukemic K562 cells was induced by the glutathione-depleting agent, diethylmaleate (DEM). We have shown for the first time that proteasomes isolated from the nuclei of control and apoptotic K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. As well, the trypsin-and chymotrypsin-like activities of nuclear proteasomes and the specificity of proteasomal nucleolysis of several individual messenger RNAs (c-fos and c-myc) were found to change under DEM action in K562 cells. DEM treatment of K562 cells led to a modification of proteasomal zeta/α5 and iota/α6 subunits associated with RNase activity. The obtained results argue in favor of changes of proteasomal subunit composition, phosphorylation state, and enzymatic activities, i.e., indicate the so-called reprogramming of the nuclear proteasome population during induced apoptosis in K562 cells.

Live Cell Imaging of Outward and Inward Vesiculation Induced by the Complement C5b-9 Complex

Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 mum) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.