[Recombinant adenovirus of microRNA-193b inhibits proliferation of K562 cells].

OBJECTIVE: To construct a recombinant adenovirus vector containing human pre-miR-193b and investigate its effect on the proliferation of chronic myelocytic leukemia K562 cells. METHODS: The cDNA of pre-miR-193b was obtained by chemical synthesis and inserted into the adenoviral shuttle vector pAdTrack-CMV. The recombinant shuttle plasmid was linearized by Pme I and transformed into AdEasier cells for homologous recombination in E.coli BJ5183 with the adenoviral backbone plasmid pAdEasy-1. The recombinant adenoviral plasmid was linearized by Pac I and then used for transfecting HEK293 cells. After package and amplification in HEK293 cells, the virus titer was determined by serial dilution assay. The expression level of miR-193b in K562 cells was detected by real-time quantitative PCR. The cell proliferation was observed by MTT assay. RESULTS: The recombinant plasmid named pAd-miR-193b was confirmed by restriction enzyme analysis and sequencing. The recombinant adenovirus could infect K562 cells efficiently. Compared with control group, real-time quantitative PCR revealed that the expression of miR-193b was significantly up-regulated in K562 cells infected with pAd-pre-miR-193b, and the cell proliferation was suppressed significantly in K562 cells with up-regulated miR-193b expression. CONCLUSION: The over-expression of miR-193b may suppress the proliferation of K562 cells.

[Over-expression of neutrophil elastase promotes proliferation and inhibits apoptosis in K562 cells].

OBJECTIVE: To establish recombinant adenovirus carrying human neutrophil elastase (NE) gene using AdEasy system, over-express NE in K562 cell line and observe the effects of NE on K562 cell proliferation and apoptosis. METHODS: NE gene was amplified with RNA extracted from acute premyelocytic leukemia (APL) HL-60 cells as a template using reverse transcription-PCR. The coding sequence was cloned into shuttle plasmid pAdTrack-CMV to obtain the recombinant plasmid named pAd-NE. After digested with HindIII and EcoRV and sequenced, the pAd-NE was transformed to competent E.coli BJ5183 containing adenovirus backbone plasmid pAdEasy-1. The obtained recombinant adenovirus plasmid Ad-NE was digested with PacI and transfected into AD293 cells for packaging. Fourteen days later, primary recombinant adenovirus Ad-NE was harvested, and then subjected to five cycles of amplification, titer determination and PCR identification. K562 cells were infected by the recombinant adenovirus. The infection efficiency was observed under a fluorescence microscope and detected by flow cytometry. Western blotting was used to detect NE expression. The proliferation of K562 cells was detected by CCK-8 assay. Cell cycle and apoptosis was measured by annexin V/PI accompanied by flow cytometry. RESULTS: HindIII and EcoRV digestion and sequencing suggested that the recombinant vector Ad-NE was successfully constructed. The recombinant plasmid Ad-NE was packaged in AD293 cells as expected. Following five-cycle amplification, the viral titer was up to 1.64 × 10¹² pfu/mL. GFP expression observed by fluorescence microscopy and flow cytometry implied that the infection efficiency of Ad-NE in K562 cells reached about 80%. Western blotting showed that NE expression was up-regulated in K562 cells. CCK-8 assay revealed that the proliferation of K562 cells over-expressing NE was enhanced. Meanwhile, flow cytometry indicated that the K562 cells were arrested in S phase and the apoptosis rate was highly reduced. CONCLUSION: Over-expressed NE in K562 leukemia cells could promote cell proliferation, inhibit apoptosis and block cell cycle in S phase.

Massive splenomegaly in acute erythroid leukaemia (FAB Class-M6): an unusual presentation.

AML-M6 has a peak incidence in the seventh decade with slight male preponderance, and can also present at a younger age. The usual features are anaemia, thrombocytopenia, malaise, fatigue, easy bruising, epistaxis and petechiae. Splenomegaly may occur in 20-40 % of the cases but massive splenomegaly is rare presentation and have been only reported once in humans and once in animals. A 22 year Asian female, presented with fatigue, pallor, mild jaundice, exertional dyspnoea, epigastric pain, tender right hypochondrium and massive splenomegaly. Investigations revealed anaemia and thrombocytopenia, tear drop cells, basophilic stippling, piokilocytosis and anisochromia; increased uric acid and LDH. Abdominal ultrasound showed enlarged liver (22cm) and spleen (20cm). Bone marrow aspiration revealed 51% erythroid and 24% non-erythroid precursors, depressed leukopoeisis and megakarypoeisis. Erythroblasts were PAS and CD71 positive and also reacted to Antihaemoglobin-Antibody. This report highlights characteristic features and diagnostic criteria of erythroleukaemia, differential diagnosis of massive splenomegaly and their rare association.

Retrovirus-transformed erythroleukemia cells induce central nervous system failure in a new syngeneic mouse model of meningeal leukemia.

Lack of suitable mouse models for central nervous system (CNS)-associated leukemias has hindered mechanism-guided development of therapeutics. By transplanting retrovirus-transformed mouse erythroleukemia cells into syngeneic mice, we developed a new animal model of meningeal leukemia associated with rapid paralysis. Necropsy revealed massive proliferation of the leukemic cells in the bone marrow (BM) followed by pathological angiogenesis and invasion of the leukemic cells into the meninges of the CNS. Further analysis demonstrated that the erythroleukemia cells secreted high levels of VEGF and preferentially adhered in vitro to fibronectin. This unique animal model for meningeal leukemia should facilitate studies of engraftment and proliferation of leukemic cells in the BM and their invasion of the CNS as well as pre-clinical evaluation of experimental therapeutics for CNS-associated leukemias.

On the nature of the tumor-initiating cell.

Certain aspects of tumors that may influence areas of basic biology and medicine are reviewed. The hypothesis that malignant stem cells evolve from normal stem cells, is considered. Information is being accumulated on the possibility that certain cell populations that can be propagated as cell lines in vitro can produce cells with features of differentiated cells in addition to others that maintain the line and, in some cases may also initiate tumor formation in vivo. Up to the present time, there is evidence to show that cancer stem cells persist in many cell lines. Tyrosine kinase inhibition produces combinations of autophagy and apoptosis in the human erythroleukemia cell line TF-1 hinting at a heterotypic aggregation of cells containing cancer stem cells. Finally, the mechanisms of cancer development, invasion and metastasis are operatively defined. The purpose of this paper is to review some of the salient features of cancer stem cells in support of the proposal that research in neoplasia be increased. Rather than presenting details of various studies, we have attempted to indicate general areas in which work has been done or is in progress. It is hoped that this survey of the subject will demonstrate a variety of opportunities for additional research in human neoplasia.

[TF-1 cell apoptosis-inducing effect of matrine and its effect on SALL4 expression].

OBJECTIVE: To explore the mechanism of matrine (Mat) induced human erythroleukemia TF-1 cell apoptosis and its effect on SALL4 expression. METHOD: Different concentrations of the Mat (0.5, 1.0, 1.5, 2.0 g x L(-1) ) were cultured in vitro in TF-1 cells at different time (24, 48, 72 h). Cell proliferation was assayed by MTT. Cell cycle was determined by flow cytometry (FCM). Cell apoptosis was detected by Annexin V and PI double staining method. SALL4 mRNA expression was detected by reverse transcription RT-PCR (RTT-PCR). RESULT: Administrated with Mat (0.5-2.0 g x L(-1)) after 24, 48, 72 h, the proliferation of TF-1 cells were inhibited (P < 0.01) , and in dose- and time-dependent manner. Half inhibitory concentration (IC50 ) was 1.0 g L(-1) at 48 h. After 48 h that the Mat acted on TF-1 cells, the proportion of G0/G1 phase cells increased while compared with the control group, and S phase cells decreased (P < 0.01). Apoptosis were 8.6% , 11.21%, 15.26% , 17.63%, which showed statistically significant difference (P < 0.01) compared with the control group (5.05%). RT-PCR results showed the ratio between SALL4 mRNA expression and beta-actin (internal reference) expression significantly decreased (P < 0.01) with Mat dose increased. CONCLUSION: In a certain range of concentration and time, Mat can inhibit TFT-1 cells proliferation. The mechanism is to make the cells G0/G1 phase blocked, to inhibit SALL4 gene expression and induce cell apoptosis.

Induction of erythroid differentiation in human erythroleukemia cells by depletion of malic enzyme 2.

Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel metabolic target for leukemia therapy.

Aurintricarboxylic acid inhibits protein synthesis independent, sanguinarine-induced apoptosis and oncosis.

Sanguinarine, a benzophenanthridine alkaloid, has anticancer potential through induction of cell death. We previously demonstrated that sanguinarine treatment at a low concentration (1.5 microg/ml) induced apoptosis in K562 human erythroleukemia cells, and a high concentration (12.5 microg/ml) induced the morphology of blister formation or oncosis-blister cell death (BCD). Treatment of cells at an intermediate sanguinarine concentration (6.25 microg/ml) induced diffuse swelling or oncosis-diffuse cell swelling (DCS). To assess the underlying mechanism of sanguinarine-induced apoptosis and oncosis-BCD in K562 cells, we studied their response to pre-treatment with two chemical compounds: aurintricarboxylic acid (ATA) and cycloheximide (CHX). The pretreatment effects of both chemical compounds on apoptosis and oncosis-BCD were evaluated by measuring multiple parameters using quantitative morphology, electron microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling and annexin-V-binding. ATA, a DNA endonuclease inhibitor, efficiently prevented DNA nicking and inhibited apoptosis almost completely and oncosis-BCD by about 40%, while CHX, a protein synthesis inhibitor, failed to inhibit both apoptosis and oncosis-BCD. These results demonstrate, first, the importance of endonuclease in sanguinarine-induced apoptosis and to some extent in oncosis-BCD and, second, that this inhibition does not require de novo protein synthesis.

Exogenous wild-type p53 gene improved survival of nude mice injected with murine erythroleukemia cell line through amelioration of hemorheological changes.

OBJECTIVES: Previous investigations have shown that human wild-type p53 gene (WTp53) inhibits the growth of leukemia and tumor cells in vitro. In the present study, the authors used nude mice and examined the therapeutic role of p53 gene for erythroleukemia in vivo in the absence of MHC effects. METHODS: The nude mice were injected with murine erythroleukemia cells (MEL), MEL cells transfected with wild-type p53 gene (MEL-W), and MEL cells transfected with mutated p53 gene (MEL-M). Abnormalities were found in the hemorheological and biophysical properties of red blood cells in all 3 groups of animals, but the abnormalities were lesser in degree and later in appearance in MEL-W group than in MEL and MEL-M groups. Furthermore, the nude mice in MEL-W group lived longer than those in MEL and MEL-M groups. RESULTS: The results showed that WTp53 restrained the growth of erythroleukemia cells in vivo and reduced the erythroleukemia tumorigenesis in the microcirculation by improving the hemorheological and biophysical properties of MEL cells, which helped to prolong the life span of nude mice suffering from erythroleukemia. CONCLUSION: These results contribute to our knowledge on the use of wild-type p53 gene for the treatment of erythroleukemia disease.

Matrine upregulates the cell cycle protein E2F-1 and triggers apoptosis via the mitochondrial pathway in K562 cells.

Matrine is a major component of Sophora Flavescens and has been reported to stimulate differentiation of erythroleukemia cells. Here we show that matrine inhibits cell proliferation or induces apoptosis in a cell type-specific manner. The latter effect was investigated in more detail in the p53 deficient erythroleukemia cell line, K562. Matrine exposure induced apoptosis in a time- and dose-dependent manner in these cells. Interestingly, co-treatment with etoposide potentiated apoptosis. Further analysis of matrine-induced apoptotic changes revealed that E2F-1 and Apaf-1 were upregulated, whereas Rb was downregulated after 24 h of exposure. This was followed by Bax translocation, cytochrome c release, and caspase-9 and -3 activation. These results demonstrate that matrine triggers apoptosis of K562 cells primarily through the mitochondrial pathway and that matrine is a potential anti-tumor drug.

Temporal variation in the expression of the p53 protein in proliferating and differentiating murine erythroleukaemia cells.

Temporal changes in the expression of p53 were investigated during hexamethylene bisacetamide (HMBA)-induced differentiation of murine erythroleukaemic (MEL) cells. Cell preparations were analysed by SDS-PAGE and western immunoblotting, which detected an immunospecific band of molecular mass 53 kDa. This analysis provided semi-quantitative information. Cell extracts were analysed further by means of ELISA techniques, using a p53-specific antibody, to provide quantitative data. In time series experiments in which cells were isolated at 15, 30 and 60 min intervals, dynamic variations in the expression of the p53 protein were detected in both the untreated and the HMBA-treated MEL cells. In all cases, the effects were complex with variations in amplitude, frequency and phasing of the rhythms. The available evidence suggests that the observed patterns, like periodic variations in other systems, are modified in rhythmic fashion with respect to period and amplitude. The results add further support for our view that it is essential to consider the dynamics when interpreting the role of p53 and cellular processes in general.

Spi-1/PU.1 participates in erythroleukemogenesis by inhibiting apoptosis in cooperation with Epo signaling and by blocking erythroid differentiation.

Overexpression of the transcription factor Spi-1/PU.1 in mice leads to acute erythroleukemia characterized by a differentiation block at the proerythroblastic stage. In this study, we made use of a new cellular system allowing us to reach graded expression of Spi-1 in preleukemic cells to dissect mechanisms of Spi-1/ PU-1 in erythroleukemogenesis. This system is based on conditional production of 1 or 2 spi-1-interfering RNAs stably inserted into spi-1 transgenic proerythroblasts. We show that Spi-1 knock-down was sufficient to reinstate the erythroid differentiation program. This differentiation process was associated with an exit from the cell cycle. Evidence is provided that in the presence of erythropoietin (Epo), Spi-1 displays an antiapoptotic role that is independent of its function in blocking erythroid differentiation. Apoptosis inhibited by Spi-1 did not involve activation of the Fas/FasL signaling pathway nor a failure to activate Epo receptor (EpoR). Furthermore, we found that reducing the Spi-1 level yields to ERK dephosphorylation and increased phosphorylation of AKT and STAT5, suggesting that Spi-1 may affect major signaling pathways downstream of the EpoR in erythroid cells. These findings reveal 2 distinct roles for Spi-1 during erythroleukemogenesis: Spi-1 blocks the erythroid differentiation program and acts to impair apoptotic death in cooperation with an Epo signaling.

Activation of Prn-p gene and stable transfection of Prn-p cDNA in leukemia MEL and neuroblastoma N2a cells increased production of PrP(C) but not prevented DNA fragmentation initiated by serum deprivation.

Prion protein (PrP(C)) via its isoform PrP(SC) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). We observed that murine erythroleukemia (MEL) cells arrested in phase G(1) undergo transcriptional activation of Prn-p gene. Here, we explored the potential role of activation of Prn-p gene and cytosolic accumulation of PrP(C) in growth arrest, differentiation, and apoptotic DNA fragmentation by stably transfecting MEL and N2a cells with Prn-p cDNA. Stably transfected MEL cells (clones # 6, 12, 20, 38, and 42) were assessed for growth and differentiation, while clones N2a13 and N2a8 of N2a cells for growth and apoptosis by flow cytometry using Annexin V and propidium iodide (PI). Our results indicate that (a) Induction of terminal differentiation of stably transfected MEL cells led to growth arrest, activation of Prn-p gene, concomitant expression of transfected Prn-p cDNA, suppression of bax gene, cytosolic accumulation of PrP(C), and DNA fragmentation. The latter was also induced in non-differentiated MEL cells growing under serum-free conditions; (b) similarly, serum deprivation promoted growth arrest, apoptosis/necrosis associated with DNA fragmentation in parental N2a and N2a13 cells that produced relative high level of PrP(C) and not PrP(SC). These data indicate that activation of Prn-p gene and expression of transfected Prn-p cDNA in cells of both hematopoietic and neuronal origin occurred concomitantly, and led to cytosolic accumulation of PrP(C) and DNA damage induced by serum deprivation. PrP(C) production failed to protect DNA fragmentation induced by serum deprivation. The question how does PrP(C) contribute to growth arrest and DNA fragmentation is discussed.

Radiation-induced bystander effect: activation of signaling molecules in K562 erythroleukemia cells.

Gap junction independent signaling mechanism was investigated using K562 human erythroleukemia cells. They were exposed to 2, 5, or 10 Gy of (60)Co gamma irradiation, the medium isolated 20 min post-irradiation and added to fresh cells. Evidence of radiation-induced bystander effect was observed wherein there was activation of p21, nuclear factor-kappaB (NF-kappaB), Bax, Bcl-2 and cleavage of poly(ADP-ribose) polymerase in bystander cells. The study implicates the involvement of signaling molecules released into the medium and factors like stable free radicals that are generated in the surrounding medium. The response elicited appears to be primarily via NF-kappaB and p21 activation.

Megakaryocyte cell sorting from diosgenin-differentiated human erythroleukemia cells by sedimentation field-flow fractionation.

Anticancer differentiation therapy could be one strategy to stop cancer cell proliferation. Human erythroleukemia (HEL) cell line, incubated with 10 microM diosgenin, underwent megakaryocytic differentiation. Thus, the association diosgenin/HEL could be used as a model of chemically induced cellular differentiation and anticancer treatment. The goal of this work was to determine the capacity of sedimentation field-flow fractionation (SdFFF) to sort megakaryocytic differentiated cells. SdFFF cell sorting was associated with cellular characterization methods to calibrate specific elution profiles. As demonstrated by cell size measurement methods, cellular morphology, ploidy, and phenotype, we obtained an enriched, sterile, viable, and functional fraction of megakaryocytic cells. Thus, SdFFF is proposed as a routine method to prepare differentiated cells that will be further used to better understand the megakaryocytic differentiation process.

MTB, the murine homolog of condensin II subunit CAP-G2, represses transcription and promotes erythroid cell differentiation.

Chromosome condensation is essential for proper segregation of duplicated sister chromatids in mitosis. Mammalian erythroid maturation is also associated with gradual nuclear condensation. However, few proteins that are directly involved in chromosome condensation during erythropoiesis have been identified. In this report, we show that MTB (more than blood), which was initially isolated in a yeast two-hybrid screen for proteins that interact with the basic helix-loop-helix (bHLH) protein stem cell leukemia (SCL), and later identified as the murine homolog of the condensin II subunit CAP-G2, participates in erythroid cell development. MTB interacts with SCL and another hematopoietic bHLH protein, E12, and is recruited to the nucleus by SCL and E12. In addition, MTB can repress SCL/E12-mediated transcriptional activation. Consistent with the model that MTB may function together with SCL/E12 heterodimer during erythroid cell development, MTB is highly expressed in the erythroid lineage and is upregulated upon erythroid differentiation. Moreover, overexpression of MTB promotes the terminal differentiation of the murine erythroleukemia erythroid cell line. Together, these findings demonstrate that the condensin II subunit MTB/mCAP-G2 plays a novel function during erythropoiesis and suggest that key hematopoietic transcription factors such as SCL and E12 may regulate the terminal differentiation of hematopoietic cells through the interaction with condensin complexes.

Activated Jak2 with the V617F point mutation promotes G1/S phase transition.

Hematopoietic stem cells in myeloproliferative diseases mostly retain the potential to differentiate but are characterized by hyper-responsiveness to growth factors, as well as partial factor-independent growth. The V617F activating point mutation in Jak2 has recently been associated with myeloproliferative disorders. Using various cell line models, mechanisms that contribute to Jak2V617-mediated signaling were investigated. Treatment of the Jak2V617F mutant-expressing erythroid leukemia cell line HEL with a small molecule Jak2 inhibitor was associated with a dose-dependent G(1) cell cycle arrest. This inhibition correlated with decreased expression of cyclin D2 and increased expression of the cell cycle inhibitor p27(Kip). Inhibition of Jak2V617F with a Jak2-targeted small interfering RNA approach resulted in a similar phenotype. Mechanisms leading to altered p27(Kip) and cyclin D2 likely involve inhibition of STAT5, a major target of Jak2 in hematopoietic cells, because a constitutively active form of STAT5 reduced p27(Kip) and increased cyclin D2 expression. Jak2V617F and constitutively active STAT5 also induced high levels of reactive oxygen species, which are sufficient to promote G(1)/S phase transition. In contrast, treatment of HEL cells with the antioxidant N-acetylcysteine decreased cell growth or expression of cyclin D2 and increased expression of p27(Kip). Similar results were obtained in BaF3 cells transfected with Jak2V617F, but these cells required coexpression of the erythropoietin receptor for optimal signaling. These results suggest that regulation of cyclin D2 and p27(Kip) in combination with redox-dependent processes promotes G(1)/S phase transition downstream of Jak2V617F/STAT5 and therefore hint at potential novel targets for drug development that may aid traditional therapy.

Phosphatidylinositol 4-phosphatase type II is an erythropoietin-responsive gene.

The erythroleukemia developed by spi-1/PU.1 transgenic mice is a multistep process. At disease onset, preleukemic cells are arrested in differentiation at the proerythroblast stage (HS1 stage) and their survival and growth are under the tight control of erythropoietin (Epo). During disease progression, malignant proerythroblasts characterized by Epo autonomous growth and in vivo tumorigenicity can be isolated (HS2 stage). During analysis of transcriptional profiling representive of discrete stages of leukemic progression, we found that the phosphatidylinositol 4-phosphatase type II gene was turned off in malignant cells. PI-4-phosphatase II is an enzyme that hydrolyses the 4-phosphate position of phosphatidylinositol-3-4-bisphosphate (PtdIns(3,4)P(2)) to form PtdIns(3)P. Using malignant cells engineered to stably express PI-4-phosphatase II, we showed that PI-4-phosphatase II reduced Akt activation level. Moreover, stimulation of malignant cells with Epo-induced PI-4-phosphatase II transcription pointing this gene as an Epo-responsive gene. This study provides first insight for a physiological role of PI-4-phosphatase II in the proerythroblast by controlling Epo responsiveness through a negative regulation of the PI3K/Akt pathway.

The effect of non-specific interactions on cellular adhesion using model surfaces.

The contribution of non-specific interactions between cells and model functional surfaces was measured using a spinning disc apparatus. These model functional surfaces were created using self-assembled monolayers (SAM) of alkylsilanes terminated with epoxide, carboxyl (COOH), amine (NH(2)), and methyl (CH(3)) groups. These SAMs were characterized using ellipsometry, atomic force microscopy, contact angle goniometry, and X-ray photoelectron spectroscopy to confirm the presence of well-formed monolayers of expected physicochemical characteristics. All substrates also demonstrated excellent stability under prolonged exposure (up to 18 h) to aqueous conditions. The adhesion strength of K100 erythroleukemia cells to the functional substrates followed the trend: CH(3) < COOH approximately epoxide << NH(2). The NH(2) SAM surface exhibited nearly an order of magnitude greater adhesion strength than the other SAMs and this non-specific effect exceeded the adhesion measured when RGD tri-peptides were also immobilized on the surface. These findings illustrate the importance of substrate selection in quantitative studies of peptide-mediated cellular adhesion.