Overexpression of transglutaminase 2 accelerates the erythroid differentiation of human chronic myelogenous leukemia K562 cell line through PI3K/Akt signaling pathway

Transglutaminase 2 (TG2) is a GTP-binding protein with transglutaminase activity. Despite advances in the characterization of TG2 functions and their impact on cellular processes, the role of TG2 in Human chronic myelogenous leukemia K562 cell line is still poorly understood. To understand the biological significance of TG2 during the differentiation of K562 cells, we established and characterized K562 cells that specifically express TG2. Non-transfected K562 cells showed the increase of membrane-bound-TG2 level after 3 days in the response to Hemin and all trans-retinoic acid (tRA), indicating that membrane recruitment of TG2 is occurred during the erythroid differentiation. However, membrane recruitment of TG2 in TG2-transfected cells revealed within earlier time period, compared with that in vector-transfected cells. The ability of membrane-bound-TG2 to be photoaffinity-labeled with [alpha-32P]GTP was also increased in TG2-transfected cells. TG2-transfected cells activated Akt phosphorylation and inactivated ERK1/2 phosphorylation, compared with vector-transfected cells. Furthermore, phosphorylation of CREB, one of the Akt substrates, was increased in TG2-transfected cells and this phenomenon was confirmed by RT-PCR analysis of several marker genes related with erythroid lineage in the absence of PI3K specific inhibitor, Wortmannin, indicating that PI3K/Akt signaling pathway also involved in the differentiation of the cell. Finally, as results of benzidine positive staining as well as hemoglobinization analysis, overexpression of TG2 revealed acceleration of the erythroid differentiation of K562 cells. Taken together, there was no increased TG2 expression level in the response of Hemin/tRA and delayed differentiation in vector transfected cells than in TG2-transfected cells, suggesting that suppression of TG2 expression may retard the erythroid differentiation of K562 cells. Therefore, our study may give a new insight for another aspect of the development of this disease.     

Induction of megakaryocytic differentiation in chronic myelogenous leukemia cell K562 by 3-hydrogenkwadaphnin

3-Hydrogenkwadaphnin (3-HK) is a daphnane-type diterpene ester isolated from Dendrostellera lessertii (Thymelaeaceae) with high differentiation and apoptotic potency in leukemic cells without any measurable adverse effects on normal cells (Moosavi et al., 2005b). In this study, we report that 3-HK (12 nM) has the ability to cease proliferation, induce differentiation and apoptosis in chronic myelogenous leukemia (CML) K562 cell line. The treated cells lost erythroid properties and differentiated along the megakaryocytic lineage based on the morphological features apparent after Wright-Giemsa staining, DNA content analysis and the expression of cell surface marker glycoprotein IIb as analyzed by flow cytometry. Moreover, using Hoechst 33258 and Annexin V double staining indicated the occurrence of apoptosis among the treated cells. On the other hand, restoration of the depleted GTP pool size by exogenous addition of guanosine (50 microM) reduced the effect of the drug regarding the extent of differentiation while no further enhancement of 3-HK effect was obtained by addition of exogenous hypoxanthine (100 microM). These interesting results necessitate further investigation regarding the mechanism of action of this unique anti-leukemic agent.     

Lapatinib induces autophagy, apoptosis and megakaryocytic differentiation in chronic myelogenous leukemia K562 cells

Lapatinib is an oral, small-molecule, dual tyrosine kinase inhibitor of epidermal growth factor receptors (EGFR, or ErbB/Her) in solid tumors. Little is known about the effect of lapatinib on leukemia. Using human chronic myelogenous leukemia (CML) K562 cells as an experimental model, we found that lapatinib simultaneously induced morphological changes resembling apoptosis, autophagy, and megakaryocytic differentiation. Lapatinib-induced apoptosis was accompanied by a decrease in mitochondrial transmembrane potential and was attenuated by the pancaspase inhibitor z-VAD-fmk, indicating a mitochondria-mediated and caspase-dependent pathway. Lapatinib-induced autophagic cell death was verified by LC3-II conversion, and upregulation of Beclin-1. Further, autophagy inhibitor 3-methyladenine as well as autophagy-related proteins Beclin-1 (ATG6), ATG7, and ATG5 shRNA knockdown rescued the cells from lapatinib-induced growth inhibition. A moderate number of lapatinib-treated K562 cells exhibited features of megakaryocytic differentiation. In summary, lapatinib inhibited viability and induced multiple cellular events including apoptosis, autophagic cell death, and megakaryocytic differentiation in human CML K562 cells. This distinct activity of lapatinib against CML cells suggests potential for lapatinib as a therapeutic agent for treatment of CML. Further validation of lapatinib activity in vivo is warranted.     

Gamma-linolenic acid induces apoptosis and lipid peroxidation in human chronic myelogenous leukemia K562 cells

Various polyunsaturated fatty acids, especially gamma-linolenic acid (GLA), inhibit the growth of a variety of tumor cells. Some evidence indicates that polyunsaturated fatty acid can kill cells by apoptosis. In the current study, we tested the apoptotic effect of GLA on human chronic myelogenous leukemia K562 cells. GLA induced K562 cell death in a dose-dependent manner. Typical apoptotic nuclei were shown by staining of K562 cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. Flow cytometric analysis also demonstrated that GLA caused dose-dependent apoptosis of K562 cells. The apoptosis could be inhibited by a pancaspase inhibitor (z-VAD-fmk), suggesting the involvement of caspases. Further, release of cytochrome c, activation of caspase-3 and cleavage of PARP were found in GLA-induced apoptosis. GLA treatment could also elevate lipid peroxidation in K562 cells, and antioxidant α-tocopherol could reverse the cytotoxicity of GLA. The saturated fatty acid SA, which did not exhibit significant increase in lipid peroxidation, also did not induce cytotoxicity. Intracellular GSH was also determined, and there was no marked change of GSH levels in cells after incubation with GLA compared with the control. These results demonstrate that GLA could induce apoptosis in K562 cells. Apoptosis is mediated by release of cytochrome c, activation of caspase-3. Lipid peroxidation may play a role in GLA cytotoxicity.     

Calcitonin receptor gene expression in K562 chronic myelogenous leukemic cells

Background  

The 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.     

Induction of erythroid differentiation of human K562 cells by 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives

In this paper we report the synthesis of twelve 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives and the results obtained on their effects in inducing erythroid differentiation of human leukemic K562 cells. The data obtained demonstrate that two of the newly synthetized compounds are able to induce erythroid differentiation of K562 cells. In addition, these same compounds potentiate K562 erythroid differentiation induced by cytosine arabinoside, retinoic acid and mithramycin. Inducers of erythroid differentiation stimulating fetal gamma-globin synthesis could be considered for possible use in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes.     

Fluctuation of gene expression for poly(ADP-ribose) synthetase during hemin-induced erythroid differentiation of human leukemia K562 cells and its reversion process

We have studied the regulation of gene expression for poly(ADP-ribose) synthetase during erythroid differentiation and its reversion process. When human leukemia K562 cells were incubated in the presence of 80 microM hemin, benzidine-positive cells appeared at day 2 and 90% of the cells became positive at day 6. However, RNA blot analysis reveals that mRNA for gamma-globin was already abundant in untreated K562 cells and the level of the message was slightly increased by hemin-treatment. Spectroscopic analysis and polyacrylamide gel electrophoresis of the induced cell extracts indicate that hemoglobin molecules were not detected in untreated cells, and increased successively up to day 6. The hemin-induced cells were thoroughly washed, and then recultured in the absence of hemin. The benzidine-positive cells mostly disappeared 3 days after the elimination of the inducer. During the hemin-induced erythroid differentiation, the activity and mRNA for poly(ADP-ribose) synthetase decreased to 50% and 20% of the initial level at day 3 and a low level of the gene expression was maintained afterwards, whereas the activity and mRNA returned to the initial value 1 day after hemin elimination. The results indicate that the hemin-induced erythroid differentiation of K562 cells is a reversible process and depression of the synthetase may be involved in the progress of differentiation.     

Studies of purine and tiazofurin metabolism in drug sensitive human chronic myelogenous leukemia K 562 cells

Antineoplastic activity of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is mediated by an anabolite of the drug thiazole-4-carboxamide adenine dinucleotide (TAD), an analog of NAD which inhibits IMP dehydrogenase activity resulting in the depletion of guanylate pools and cell death. Human chronic myelogenous leukemia K 562 cells were found to be sensitive to tiazofurin with an IC50 of 19.2 microM. TAD content in K 562 cells (1.3 nmol/10(9)/h) was in the range found in susceptible murine and human tumor cells. Studies were conducted to relate tiazofurin toxicity with biochemical effects by examining nucleotide pools. Among the nucleotides, only guanylate pools were significantly depleted by the drug. To further study the effect of the drug on the purine nucleotide de novo and salvage biosynthetic pathways, flux of radiolabelled formate and guanine was employed. The results showed that de novo synthesis of guanylates was curtailed primarily by the drug's action without influencing adenylate biosynthesis or salvage of guanine to guanylates. These studies show that K 562 cells are sensitive to selective inhibition of de novo guanylate pathway indicating that human chronic myelogenous leukemia in blast crisis might be a good candidate for Phase II clinical trials with tiazofurin.     

Sustained mitogenic effect on K562 human chronic myelogenous leukemia cells by dietary lectin,jacalin

Dietary lectins have been shown to affect the proliferation of human cancer cell lines. The anti-proliferative effects of lectins from varied sources have been extensively studied and in some cases, the underlying mechanism has been explored. Except for peanut agglutinin (PNA), the mitogenic effects of no other lectins have been studied in detail. In the present study, we have shown that jacalin, lectin purified from jackfruit (Artocarpus integrifolia) seeds act as a mitogen for K562, the Bcr-Abl expressing erythroleukemia cell line (K562) and the effect was found to be dose dependent. K562 cells remained in the proliferative state for a longer period even after the withdrawal of jacalin stimulation, thus jacalin was found to induce sustained mitogenic effect on K562 cells. Further, conditioned media from K562 cells treated with jacalin were observed to have the similar mitogenic effect even in the presence of galactose. Importantly, galactose which is a known ligand for jacalin will interact with functionally active jacalin present in the conditioned media and neutralise its effect. In addition, jacalin treatment also resulted in increased mRNA expression levels of pro-inflammatory cytokines including IL-1β, IL-6 and IFN-γ. Our results indicate that jacalin induces secretion of soluble molecules, which maybe responsible for this observed increased proliferation of K562 cells.     

Involvement of nPKC-MAPK pathway in the decrease of nucleophosmin/B23 during megakaryocytic differentiation of human myelogenous leukemia K562 cells

Human myelogenous leukemia K562 cells were induced to undergo megakaryocytic differentiation by long-term treatment with phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The protein level of nucleophosmin/B23 (NPM/B23), a nucleolar protein, was substantially decreased upon TPA treatment. In this study, we found that the proteasome inhibitors blocked the decrease of NPM/B23 protein in response to TPA, suggesting the proteasomes were involved in the downregulation of NPM/B23 upon megakaryocytic differentiation. To investigate the signaling pathway in the downregulation of NPM/B23 during early TPA-induced megakaryocytic differentiation of K562 cells, K562 cells were treated with TPA in the presence of the PKC isozyme-selective inhibitors, GF109203X and Gö 6976, or MEK1 inhibitor, PD98059. The decrease of NPM/B23 protein in the TPA-treated K562 cells was blocked by GF109203X but not by Gö 6976, suggesting the involvement of novel PKCs in the downregulation of NPM/B23 during TPA-induced megakaryocytic differentiation of K562 cells. The application of MEK1 inhibitor PD98059 upon TPA treatment blocked the TPA-induced decrease of NPM/B23 protein and aborted the megakaryocytic differentiation but not to break through the cell growth arrest. Unlike NPM/B23, the degradation of nucleolin in the TPA-treated K562 cells could not be blocked by PD98059 while the TPA-induced megakaryocytic differentiation was abrogated. The decrease of NPM/B23 protein seems to be more correlated with the novel PKC-MAPK-induced megakaryocytic differentiation than another nucleolar protein, nucleolin. Taken together, our results indicated that novel PKC-MAPK pathway was required for the decrease of NPM/B23 during TPA-induced megakaryocytic differentiation.     

A novel kind of nitrogen heterocycle compound induces apoptosis of human chronic myelogenous leukemia K562 cells

The effects of a novel kind of nitrogen heterocycle compound, which was synthesized in our laboratory previously, on human chronic myelogenous leukemia K562 cells were investigated. The morphological changes were observed by Acridine orange (AO) staining. The screened results through DNA fragmentation and the Annexin V-FITC/PI staining assay showed that compound 8 blocked cell cycles at G(1) phase which led to apoptosis. The increase of caspase-3, 8, and 9 was detected, indicating that both of death-receptor and mitochondria-pathways were activated. Compound 8 induced a biphasic alteration in mitochondrial membrane potential of K562 cells. A dramatic elevation of Ca(2+) was also observed. In addition, a transient increase of ROS was also involved in the process. This study showed that compound 8 might be a potential chemopreventive agent for chronic myelogenous leukemia. It would guide our future work to synthesize more compounds derived from compound 8, which might have better effect, and to determine the target protein. Moreover, it might also provide a background mechanism for the introduction of this new type of promising therapeutic agent.     

K562 chronic myeloid leukemia cells modify osteogenic differentiation and gene expression of bone marrow stromal cells

Bone marrow (BM) microenvironment plays an important role in normal and malignant hematopoiesis. As a consequence of interaction with the leukemic cells, the stromal cells of the bone marrow become deregulated in their normal function and gene expression. In our study, we found that mesenchymal stem cells (MSC) from BM of chronic myeloid leukemia (CML) patients have defective osteogenic differentiation and on interaction with K562 CML cells, the normal MSC showed reduced osteogenic differentiation. On interaction with K562 cells or its secreted factors, MSC acquired phenotypic abnormalities and secreted high levels of IL6 through NFκB activation. The MSC derived secreted factors provided a survival advantage to CML cells from imatinib induced apoptosis. Thus, a therapy targeting stromal cells in addition to leukemia cells might be more effective in eliminating CML cells.     

Changes in diacylglycerol and cyclic GMP during the differentiation of human myeloid leukemia K562 cells

When the human myeloid leukemia cell line, K562, was induced to differentiate along the erythroid lineage by a 4 day treatment with 10 microM tiazofurin, the cellular content of diacylglycerol decreased to 35% of the value in untreated control cells. Under the same conditions the content of cGMP decreased to 61% of the control value. Tiazofurin inhibits guanine nucleotide biosynthesis and lowers cellular GTP. When guanosine and adenine were added together with tiazofurin, the differentiation of K562 was prevented, the concentration of diacylglycerol was maintained at control values, and the reduction in the concentration of cGMP was partially prevented. Other inducers of differentiation which acted by different mechanisms, caused similar changes in the concentrations of diacylglycerol and cGMP.     

慢性髓性白血病K-562细胞的凋亡耐受

慢性髓性白血病（CML）K－562细胞株对与类别无关的多种凋亡诱导剂具有耐受性,表现为凋亡潜伏期延长,caspases激活延迟及其活性谱和亚细胞分布改变,电泳不易显示典型、清晰的DNA梯形条带。K－562细胞凋亡耐受机制复杂,Bcr-Abl上调ERK／MAPK通路,经尚未阐明的中间环节,抑制凋亡诱导剂引起的线粒体释放反应,导致caspase-3激活延迟,是其可能的机制。目前研究中的克服K－562细胞凋亡耐受、治疗CML的策略包括特异性酪氨酸激酶抑制剂等。     

Emodin can induce K562 cells to erythroid differentiation and improve the expression of globin genes

In China, the traditional Chinese medicine “YiSui ShenXu Granule” has been used for treating β-thalassemia over 20 years and known to be effective in clinic. Several purified components from “YiSui ShenXu Granule” are tested in K562 cells to reveal its effect on globin expression and erythroid differentiation, and one of the purified components, emodin, was demonstrated to increase the expression of α-, ε-, γ-globin, CD235a, and CD71 in K562 cells. Moreover, the increase of their expression is emodin concentration-dependent. The mRNA and microRNA (miRNA) expression profiles are further analyzed and 417 mRNAs and 35 miRNAs with differential expression between untreated and emodin-treated K562 cells were identified. Among them, two mRNAs that encode known positive regulators of erythropoiesis, ALAS2, and c-KIT respectively, increased during emodin-induced K562 erythroid differentiation, meanwhile, two negative regulators, miR-221 and miR-222, decreased during this process. These results indicate that emodin can improve the expression of globin genes in K562 cells and also induce K562 cells to erythroid differentiation possibly through up-regulating ALAS2 and c-KIT and down-regulating miR-221 and miR-222.