Differential tyrosine phosphorylation of leukemic cells during apoptosis as a result of treatment with imatinib mesylate

Bcr-Abl fusion tyrosine kinase contributes to leukemic transformation. Imatinib mesylate inhibits Bcr-Abl tyrosine kinase, resulting in a blockage of tyrosine phosphorylation in its downstream pathways. We analyzed the alteration of tyrosine phosphorylation, on BCR/ABL+ chronic myelogenous leukemia cells, after treatment with imatinib mesylate. Data were collected using a two-dimensional gel electrophoresis followed by Western blot and mass spectrometry. The inhibition of Bcr-Abl tyrosine kinase by 2.5 microM imatinib mesylate caused both cell cycle arrest in the G0/G1 phase and increased the portion of apoptotic cells. As a result, the population of leukemic cells decreased by 30% and 70% compared to controls at 24 and 72 h, respectively. Furthermore, treatment with imatinib mesylate altered tyrosine phosphorylation of 24 protein spots as the incubation time proceeded from 0 to 24 and 72 h. Ten of the 24 protein spots are visible at all three times. Four are detectable at both the 0 and 24 h points in time. Eight were detectable only at time 0.     

Cytoprotective effect of imatinib mesylate in non-BCR-ABL-expressing cells along with autophagosome formation

Treatment with imatinib mesylate (IM) results in an increased viable cell number of non-BCR-ABL-expressing cell lines by inhibiting spontaneous apoptosis. Electron microscopy revealed an increase of autophagosomes in response to IM. IM attenuated the cytotoxic effect of cytosine arabinoside, as well as inhibiting cell death with serum-deprived culture. Cytoprotection with autophagosome formation by IM was observed in various leukemia and cancer cell lines as well as normal murine embryonic fibroblasts (MEFs). Complete inhibition of autophagy by knockdown of atg5 in the Tet-off atg5^−/− MEF system attenuated the cytoprotective effect of IM, indicating that the effect is partially dependent on autophagy. However, cytoprotection by IM was not mediated through suppression of ROS production via mitophagy, ER stress via ribophagy, or proapoptotic function of ABL kinase. Although the target tyrosine kinase(s) of IM remains unclear, our data provide novel therapeutic possibilities of using IM for cytoprotection.     

KLF1和KLF9对K562细胞红系分化的协同调控作用

Krüppel样因子(Krüppel-like factors,KLFs)是锌指蛋白超家族的一个亚家族,参与细胞内的多种生理、病理过程,该家族成员在红细胞分化发育过程中发挥非常重要的作用,但是家族成员间对红系分化的协同调控作用还鲜有报道。本课题组前期研究发现,KIF家族成员KLF1和KLF9在已分化的红系细胞中的表达水平显著高于造血干细胞。为进一步探讨二者在红系分化中是否存在协同作用,本研究在K562细胞中分别过表达/敲低表达KLF1和KLF9,检测二者表达的相关性,发现KLF1和KLF9的基因表达呈现正相关,且二者共表达可以显著促进K562细胞红系分化,特异地增强β-珠蛋白的表达。通过对KLF1、KLF9单独和共同过表达、敲低表达的K562细胞转录组数据的分析发现二者可能通过PI3K-Akt和FoxO通路协同调控红系分化,FOS、TF、IL8是协同调控的候选靶基因。本研究结果为后续深入研究KLF1和KLF9协同调控红系分化的分子机制奠定了基础。     

Effect of bacitracin on erythroid differentiation of MEL cells

Bacitracin, an antibiotic widely utilized in clinical and veterinary use, was tested on murine erythroleukemia (MEL) cells. Tests were performed to evaluate the capacity of the drug to interfere with erythroid differentiation. Cells were exposed to a single treatment in S phase at sublethal doses of bacitracin. Two responses were found depending on the drug concentration. At higher concentrations (25 g/ml and 250ng/ml) a reduction in number of differentiating cells was observed but the kinetics of the process remained unchanged. At lower concentrations (from 2.5 ng/ml to 2.5 fglml) a dramatic alteration of the dynamic of differentiation was found. These two responses are related to different activities of the DNA repair mechanisms. Higher doses of bacitracin stimulate repair while lower concentrations are not able to activate repair, as demonstrated by tests with hydroxyurea. The bacitracin-induced damage can be considered a stable genetic andlor epigenetic alteration, as demonstrated by the high frequency of mutant clones isolatedfrom low-dose treated cells. The suitability of MEL cells system in evaluating genotoxicity of drugs for veterinary use is underlined.Abbreviations MEL murine erythroleukemia - HU hydroxyurea     

Induction of erythroid differentiation by the anthracycline antitumor antibiotic pyrromycin

The oligosaccharide-anthracyclines, aclacinomycin A, marcellomycin and musettamycin, are potent inducers of erythroid differentiation in hemopoietic cell lines of rodent and human origin. The present studies revealed that pyrromycin, a closely related monosaccharide-anthracycline, induced erythroid differentiation in Friend leukemia cells and in the human leukemia cell line K 562. Pyrromycin, marcellomycin and musettamycin, which possess an identical aglycone structure containing a Cl-hydroxyl group, exhibited relatively low optimal inductive concentrations. In contrast, the optimal inductive concentration of aclacinomycin A, which lacks the Cl-hydroxyl group, was markedly higher, i.e., the differentiation inducing capacity was lower. It should be noted, however, that the yield of differentiated cells following treatment with the monosaccharide-anthracycline pyrromycin was distinctly lower than that after treatment with the oligo-saccharide-anthracyclines, aclacinomycin A, marcellomycin or musettamycin. Thus, our data indicate that the efficacy of anthracyclines to induce erythroid differentiation is related to a) the presence of a Cl-hydroxyl group in the aglycone and b) the presence of an oligosaccharide side chain.Abbreviation Hb hemoglobin     

Phorbol 12-myristate 13-acetate prevents apoptosis in erythroleukemia K562 cells induced by some nucleosides

We studied the ability of phorbol 12-myristate 13-acetat to prevent erythroid differentiation and apoptosis in erythroleukemic K562 cells induced by cytidine, thymidine, and guanosine. The exposure of cancer cells to combinations of phorbol 12-myrsitate 13-acetate (100 nM) nucleosides for two days led to a loss of hemoglobin production (marker of erythroid differentiation) in cells and increased expression of monocyte-macrophage lineage associated surface antigen CD14. The treatment of K562 cells with nucleosides only was accompanied by the activation of caspase-3 and caspase-9, rather than caspase-6, increased fluorescence of ethidium bromide and DAPI upon binding to DNA, and apoptosis. Intracellular activation of caspase-6, inhibition of caspase-9, a markedly decreased activity of caspase-3 and of fluorescence of DNA-binding dyes, and inhibition of apoptosis were observed when the cells were treated with phorbol 12-myeristet 13-acetate combined with nucleosides.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 18–25.Original Russian Text Copyright © 2005 by Volkova, Malysheva, Nemova.     

Role of ERK activation in growth and erythroid differentiation of K562 cells

Inhibition of signaling through Ras in BCR-ABL-positive pluripotent K562 cells leads to apoptosis and spontaneous differentiation. However, Ras-induced activation of the mitogen-activated protein kinase ERK has been suggested to play a critical role in either growth or differentiation in different model systems. We studied the role of ERK activation in the growth-promoting and anti-apoptotic effect of Ras and its involvement in hemin-induced nonterminal erythroid differentiation using the BCR-ABL-positive K562 cell line as a model. K562 cells were stably transfected with ERK1 or the dominant inhibitory mutant of ERK1 (ERK1-KR). Overexpression of ERK1-KR inhibited cell growth with an approximately fourfold increase in doubling time and induced apoptosis in K562 cells. Incubation with the MEK1 inhibitor UO126 inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner as well. In the presence of exogenously added hemin, K562 cells differentiate into erythroblasts, as indicated by the production of large amounts of fetal hemoglobin. We examined the activation of MAP kinases during hemin-induced differentiation. The ERK1 and 2 activity increased within 2 h post hemin treatment and remained elevated for 24-48 h. During this time, fetal hemoglobin synthesis also increases from 0.8 to 10 pg/cell. There was no activation of JNK or p38 protein kinases. The hemin-induced accumulation of hemoglobin was inhibited in ERK1-KR overexpressing cells and was enhanced in the wild-type ERK1 transfectants. Our results suggest that ERK activation is involved in both growth and hemin-induced erythroid differentiation in the BCR-ABL-positive K562 cell line.     

DAP12 ITAM motif regulates differentiation and apoptosis in M1 leukemia cells

DAP12 is an immunoreceptor tyrosine-based activation motif (ITAM)-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. DAP12 is expressed not only in NK cells, but also in myeloid cells. Previously, we reported that DAP12 was likely to be involved in monocyte differentiation to macrophage. In this study, we established the mutant DAP12-M1 transfectants (Y76F-M1) that have mutation at their ITAM motifs. We observed that Y76F-M1 cells could not differentiate to macrophages by stimulation via DAP12, whereas wild type DAP12 transfectants (FDAP-M1) could. Furthermore, we demonstrated that the apoptosis signal mediated by LPS was inhibited in Y76F-M1 cells, but was augmented in FDAP-M1 cells. In contrast to the LPS-mediated apoptosis, the combination of LPS and DAP12 stimulation showed good cell viability in FDAP-M1 cells. Collectively our studies demonstrated that DAP12 has a critical role for macrophage differentiation and LPS induced apoptosis in M1 leukemia cells.     

Autocrine signaling of platelet-derived growth factor regulates disabled-2 expression during megakaryocytic differentiation of K562 cells

Platelet-derived growth factor (PDGF) is involved in megakaryocytopoiesis and is secreted into the culture medium during megakaryocytic differentiation of human leukemic cells. We investigate whether PDGF plays a role in the regulation of the adapter protein Disabled-2 (DAB2) that expresses abundantly in platelets and megakaryocytes. Western blot analysis revealed that conditioned medium from 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated, megakaryocytic differentiating K562 cells upregulated DAB2 expression. DAB2 induction and megakaryocytic differentiation was abrogated when cells were co-treated with the PDGF receptor inhibitor STI571 or when the conditioned medium was derived from TPA-plus STI571-treated cells. Although the level of PDGF mRNA was not altered by STI571, an approximate 44% decrease in PDGF in the conditioned medium was observed. Consistent with these findings, interfering PDGF signaling by PDGF neutralization antibody or dominant negative PDGF receptors attenuated DAB2 expression. Accordingly, transfection of an expression plasmid encoding secreted PDGF upregulated DAB2. This study shows for the first time that PDGF autocrine signaling regulates DAB2 expression during megakaryocytic differentiation.     

Effect of STI-571 (imatinib mesylate) in combination with retinoic acid and gamma-irradiation on viability of neuroblastoma cells

Neuroblastoma (NB) expresses the tyrosine kinase receptors c-Kit, PDGFR-alpha and -beta-targets for STI-571.We investigated a possible combination therapy of STI-571 with retinoic acid (RA) and gamma-irradiation on NB cell viability in vitro. Expression of tyrosine kinase receptors and their ligands was examined in 6 NB cell lines by RT-PCR and FACS. The effect on cell viability was determined by MTT assay. Cell viability of all 6 NB cell lines was significantly inhibited after treatment with 20 microM STI-571 for 72h, two cell lines responding already to 10 microM. Cell lines responded irrespective of their mRNA status or cell surface expression of c-Kit, PDGFR-alpha and -beta. Co-incubation with 9-cis RA sensitized cells to the inhibitory effects of STI-571. However, pre-treatment with 9-cis RA resulted in resistance of NB cell lines to STI-571 and gamma-irradiation. Treatment of NB with STI-571 in combination with 9-cis RA might be a therapeutic strategy for patients in consolidation therapy who have completed gamma-irradiation therapy.     

Monolayer culture conditions suitable for primitive erythroid cell differentiation from embryonic stem cells in vitro

Embryonic stem (ES) cells effectively differentiated into primitive erythroid/mesodermal cells when grown in the absence of both a feeder layer and leukemia inhibitory factor (LIF). The formation of a three-dimensional structure, exogenous mesoderm induction factors and exogenous hematopoietic growth factors were not essential for their differentiation. Primitive erythroid cells were first detected on day 5 in the differentiation-permissive cultures. Differentiation into other mesodermal cells was always preceded by that into primitive erythroid cells. Precursor cells of erythroid cells but of other hematoid cells were also detected in this system. This model system is useful for studying the early steps of mesoderm formation in mouse embryogenesis.     

Study of the mechanisms of crocetin-induced differentiation and apoptosis in human acute promyelocytic leukemia cells

Crocetin, the major carotenoid in saffron, exhibits potent anticancer effects. However, the antileukemic effects of crocetin are still unclear, especially in primary acute promyelocytic leukemia (APL) cells. In the current study, the potential antipromyelocytic leukemia activity of crocetin and the underlying molecular mechanisms were investigated. Crocetin (100 µM), like standard anti-APL drugs, all-trans retinoic acid (ATRA, 10 µM) and As₂O ₃ (arsenic trioxide, 50 µM), significantly inhibited proliferation and induced apoptosis in primary APL cells, as well as NB4 and HL60 cells. The effect was associated with the decreased expressions of prosurvival genes Akt and BCL2, the multidrug resistance (MDR) proteins, ABCB1 and ABCC1 and the inhibition of tyrosyl-DNA phosphodiesterase 1 (TDP1), while the expressions of proapoptotic genes CASP3, CASP9, and BAX/BCL2 ratio were significantly increased. In contrast, crocetin at relatively low concentration (10 µM), like ATRA (1 µM) and As ₂O ₃ (0.5 µM), induced differentiation of leukemic cells toward granulocytic pattern, and increased the number of differentiated cells expressing CD11b and CD14, while the number of the immature cells expressing CD34 or CD33 was decreased. Furthermore, crocetin suppressed the expression of clinical marker promyelocytic leukemia/retinoic acid receptor-α ( PML/RARα) in NB4 and primary APL cells, and reduced the expression of histone deacetylase 1 ( HDAC1) in all leukemic cells. The results suggested that crocetin can be considered as a candidate for future preclinical and clinical trials of complementary APL treatment.     

Regulation of erythroid differentiation by miR-376a and its targets

Lineage differentiation is a continuous process during which fated progenitor cells execute specific programs to produce mature counterparts. This lineage-restricted pathway can be controlled by particular regulators, which are usually exclusively expressed in certain cell types or at specific differentiation stages. Here we report that miR-376a participates in the regulation of the early stages of human erythropoiesis by targeting cyclin-dependent kinase 2 (CDK2) and Argonaute 2 (Ago2). Among various human leukemia cell lines, miR-376a was only detected in K562 cells which originated from a progenitor common to the erythroid and megakaryotic lineages. Enforced expression of miR-376a or silencing of CDK2 and Ago2 by RNAi inhibits erythroid differentiation of K562 cells. Hematopoietic progenitor cells transduced with miR-376a showed a significant reduction of their erythroid clonogenic capacity. MiR-376a is relatively abundant in erythroid progenitor cells, where it reduces expression of CDK2 and maintains a low level of differentiation due to cell cycle arrest and decreased cell growth. Following erythroid induction, miR-376a is significantly down-regulated and CDK2 is released from miR-376a inhibition, thereby facilitating the escape of progenitor cells from the quiescent state into erythroid differentiation. Moreover, our results establish a functional link between miR-376a and Ago2, a key factor in miRNA biogenesis and silencing pathways with novel roles in human hematopoiesis.     

Identification of key genes responsible for cytokine-induced erythroid and myeloid differentiation and switching of hematopoietic stem cells by RAGE

We utilized a unique culture system to analyze the expression patterns of gene, protein, and cell surface antigen, and the biological process of the related genes in erythroid and myeloid differentiation and switching of hematopoietic stem cells （HSCs） in response to cytokine alterations. Gene-specific fragments （266） identified from five populations of cytokine-stimulated HSCs were categorized into three groups： （1） expressed specifically in a single cell population; （2） expressed in two cell populations, and （3） expressed in three or more populations. Of 145 defined cDNAs, three （2%） were novel genes. Protein two-dimensional gel electrophoresis and flow cytometry analyses showed overlapped and distinguished protein expression profiles in the cell populations studied. Biological process mapping of mRNAs expressed in erythroid and myeloid lineages indicated that mRNAs shared by both lineages attended ＇core processes,＇ whereas genes specifically expressed in either lineage alone were related to specific processes or cellular maturation. Data from this study support the hypothesis that committed HSCs （El4 or G14） cells can still be redirected to develop into myeloid or erythroid cells when erythropoietin （EPO） is replaced with granulocyte-colony stimulating factor （G-CSF） under erythroid-cultured condition or G-CSF with EPO in myeloid-cultured environment, respectively. Our results suggest that genes or proteins co-expressed in erythroid and myeloid lineages may be essential for the lineage maintenance and switching in hematopoiesis.