Epigenetic-Based Treatment Induces Apoptosis in Leukemic Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4382-4382
Author(s):  
Maria J. Carnicer ◽  
Adriana Lasa ◽  
Elena Serrano ◽  
Jorge Pena ◽  
Angel F. Remacha ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Histone Deacetylase Inhibitors ◽  
Caspase 3 ◽  
Leukemic Cell ◽  
Demethylating Agents ◽  
Leukemic Cell Lines ◽  
Wide Range

Abstract Pharmacological treatment of cancer cells with demethylating agents and histone deacetylase inhibitors synergistically reactivates the transcription of previously silenced genes. The aim of this study was to investigate the antileukemic properties of a DNA mehtyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC),an histone deacetylase inhibitor Trichostatin A (TSA) and trans-retinoc acid (ATRA), alone or in combination. The effects of these drugs on apoptosis, cell cycle progression, cell-survival pathways and restoration of proliferation-associated genes silenced by aberrant epigenetic mechanisms were assessed. Four human leukemic cell lines were used: lymphoid cell-lines REH (Tel-Aml1+) and DAUDI (c-myc) and myeloid cell-lines Kasumi-1(Aml1-Eto+) and NB4(Pml-Rara+). The cells were cultured for three days. Cell viability, percentage of apoptosis, cell cycle and apoptosis-controlling proteins were examined by multiparametric flow cytometry, western blotting and a RT-PCR low-density array (LDA)containing 48 probes. Combined 5-Aza-dC and TSA treatment induced a high degree of apoptosis and affected the cell-cycle in all the cell lines analyzed. Increased levels of cleaved PARP were detected after the double treatment and paralleled those of caspase-3 except for DAUDI cell-line where there was no cleavage of procaspase 3 suggesting that this treatment induced a death-pathway independent of caspase-3 activation. Furthermore, ATRA alone has a limited capacity to induce apoptosis in all the tested cell-lines. The genes upregulated following the combined epigenetic-active treatment in DAUDI were; PRKCG, TNFSF10, TNFRSF10B, MAPK8, CASP9, APAF1, PER1, CDKN1A, CCND1. In the REH cell line were; PRKCG, TNFRSF10A, TNFRSF10B, MAPK8, CASP8, CASP9, APAF1, CASP3, AKT1, PIK3CG, BIK, PRKCG, MCL1, CCND1, CCND2, EZH2, PER1, GCLC, GSTP1. In the KASUMI-1 cell line were; PRKCABP, TNFSF10, TNFRSF10A, TNFRSF10B, FAS, CFLAR, CASP8, APAF1, CEBPA, CCND1, CDC25C, CDKN1A, EZH2.For the NB4 cell-line were: PRKCG, PRKCABP, AKT1, TNFSF10, CFLAR, CASP8 and CASP9. We can conclude that combined treatment with demethylating agents and histone deacetylase inhibitors may be active in a wide range of human leukemias. The potential use of this combination in the commonest form of pediatric ALL warrants further investigation.

Effect of Second Mitochondria-Derived Activator of Caspases (Smac) Mimetic Compounds on Leukemic Cell Lines.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1609-1609
Author(s):  
Federica Servida ◽  
Francesco Onida ◽  
Domenico Delia ◽  
Cinzia Scavullo ◽  
Daniele Lecis ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cytotoxic Effect ◽  
Leukemic Cell ◽  
Hl60 Cell ◽  
Caspase 9 ◽  
Leukemic Cell Lines ◽  
Smac Mimetics ◽  
Smac Mimetic ◽  
Hl60 Cell Line

Abstract The apoptotic process and its dysfunctions have become the focus of extensive pharmaceutical research in solid and hematopoietic tumors as well as neurodegenerative diseases. The X-Inhibitor of Apoptosis Protein (XIAP) binds caspase 9, 3 and 7, preventing their activation and, consequently, apoptosis. The Smac/DIABLO protein, released from mitochondria, binds XIAP as a dimer on the same caspase 9 (BIR3 domain) binding site. Similarly, the Smac protein interferes with the XIAP binding site for caspases 3 and 7, thus promoting both the extrinsic and intrinsic apoptotic paths. The thin balance of this binding equilibrium is altered in various tumors, including leukemia, where XIAP is overexpressed and a caspase-dependent resistance to enter apoptosis is usually observed. Thus, XIAP inhibition via Smac mimetics’ binding is at the same time a characterized protein-protein interaction, and a validated mechanism for intervention in cancer therapy. We tested 56 Smac mimetic compounds (designed by CISI - Center for biomolecular Interdisciplinary Studies and Industrial applications of the Milan University) for their in vitro capacity to bind to the XIAP BIR3 domain. We also evaluated the ability of the Smac mimetic compounds to inhibit the growth of the human leukemia HL60, K562 and Jurkat cell lines (derived from patients with promyelocyitic leukemia, blastic phase-CML and T acute lymphoblastic leukemia, respectively). Nine compounds which were shown to be active, were further investigated for their effect on cell cycle (by DNA staining with propidium iodide and cytofluorimetric analysis) and for possible synergistic effect in combination with other chemotherapeutic drugs (Cytarabine, Etoposide and Idarubicine). The same compounds were also tested on normal CD34+ hematopoietic progenitor cells. The cytotoxicity was evaluated after 72 hours treatment with Smac mimetic compounds by a colorimetric assay for the quantification of cell proliferation and viability based on the cleavage of the WST-8 tetrazolium salt by mitochondrial dehydrogenases. The effect of Smac mimetic compounds on CD34+ cells enriched from mobilized peripheral blood was assessed as the capability of inhibiting the myeloid colony growth (CFU-GM). The data were expressed as mean percentage of 3 replicates normalized to the untreated control. Overall, a strong correlation between the binding affinity to the XIAP BIR3 domain and the cytotoxic effect on the leukemic cell lines was observed. The more promising compounds showed IC50 ranging from 0,3 to 1 microM on the HL60 cell line. The Jurkat and K562 cell lines were less sensitive, with IC50 ranging from 11,8 microM to more than 50 microM. However, in the K562 cell line, the combined treatment unveiled synergistic effect with Cytarabine and Etoposide (R Kern index = 1,4 and 1,5 respectively). No cytotoxic effect was observed on normal controls at doses up to 80 microM. A consistent sub G1 apoptotic peak (up to 53% of apoptotic cells) was observed in the HL60 cell line after 48 hrs treatment, thus suggesting a strong activation of the apoptotic process. All together, our data suggest that Smac mimetics may have a promising therapeutic potential as a new class of anticancer drugs in hematopoietic malignancies. Further experiments are currently ongoing to confirm the effectiveness of these compounds also on primary cells from leukemia patients, both as single agents and in combination with conventional drugs. In particular, due to their ability to enhance pro-apoptotic effect, Smac mimetic compounds may allow to overcome resistance of cancer cells to standard chemotherapy.

Methylation Regulates Expression of Novel Splice Variant and Wild Type Transcripts of IRF8.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3634-3634
Author(s):  
Era L. Pogosova-Agadjanyan ◽  
Hana Lee ◽  
Crystal K. Cummings ◽  
Soheil Meshinchi ◽  
Jerald P. Radich ◽  
...  
Keyword(s):  
Cell Lines ◽  
Promoter Methylation ◽  
Splice Variant ◽  
Splice Variants ◽  
Leukemic Cell ◽  
Wild Type ◽  
Over Expression ◽  
Demethylating Agents ◽  
Leukemic Cell Lines ◽  
Transcript Variants

Abstract Abstract 3634 Introduction: Interferon regulatory factor 8 (IRF8) is a transcription factor that plays a critical role in normal hematopoiesis. IRF8−/− transgenic mice develop a myeloproliferative syndrome that transforms to acute myeloid leukemia (AML). IRF8 expression varies dramatically in the blasts of AML patients. A number of biological processes, including epigenetic changes, mutations, or alternative splicing, may contribute to the variability of IRF8 expression in AML patients. We investigated the potential causes of the aberrant IRF8 expression in AML blasts. Wild-type transcript (IRF8-WT): The entire coding sequence of IRF8 was amplified using HiFi Taq polymerase and sequenced from 7 leukemic cell lines and 12 AML samples. We did not find any mutations associated with aberrant expression of IRF8. Splice Variants (IRF8-SVs): The initial studies examining IRF8 coding region suggested transcript deviation in the 5′ region. The GeneRacer kit was used to sequence 5′-capped mRNA. We identified 3 previously-undescribed transcript variants. In all 3 sequences, exon 1 was spliced out and replaced by nucleotides from the terminal end of intron 1 (Figure 1A). Some of the splice variants contained potential in-frame start codons. Expression of 5′ Splice Variants in Leukemic Cell Lines and AML Samples: We examined the expression levels of transcript variants in 12 leukemic cell lines, 246 AML samples, and hematopoietic subpopulations of cells from “healthy” adults. IRF8-SVs were expressed at significantly higher levels in some AML blasts than normal hematopoietic cells. In fact, AML cell lines with the highest IRF8 levels primarily expressed the IRF8-SVs rather than the IRF8-WT. Promoter Methylation: Interferon response element (pIRE) within the IRF8 promoter, which controls the expression of the gene, was examined by PCR after bisulfite conversion. Cell lines with very low IRF8 expression were often methylated at the pIRE locus. In addition, some cell lines with marked over-expression of IFR8-SVs also displayed methylated pIRE, suggesting that promoter methylation may also be playing a role in controlling the expression of the splice variants. pIRE methylation was also examined in a more limited number of AML samples, finding that blasts with low expression of IRF8 were methylated at this locus (Figure 1B). Reversal of Splice Variant Expression Pattern: Our previous studies suggested that methylation may play a role in controlling IRF8-SVs expression. Therefore, we examined the effect of demethylating agents on IRF8-SVs expression in U937 cells – a leukemic cell line with very high levels of IRF8-SVs. IRF8 transcripts (WT and SVs) were examined before and after exposure to therapeutic levels of 5-azacytidine (5-Aza). These studies suggested that 5-Aza exposure and subsequent demethylation of the pIRE locus was associated with restoration of the IRF8-WT and decreased IRF8-SVs expression (Figure 1C). This later data provides additional evidence that pIRE hypermethylation may regulate the expression of the novel IRF8-SVs. Conclusions: We have identified novel IRF8 transcript variants that are over-expressed in AML blasts. These novel IRF8-SVs introduce additional nucleotides at the 5′ region of the gene and may result in a new start codon. AML blasts with high levels of IRF8 transcript often over-express IRF8-SVs. Although the functional significance of these variants is unknown, methylation may play a role in regulating their expression, such that demethylating agents decrease IRF8-SVs and promote the IRF8-WT expression. Additional studies are planned to investigate the biology and clinical significance of IRF8-SVs. Initial studies suggests that over-expression of IRF8-SVs is associated with an inferior clinical outcome for adult AML patients. Disclosures: No relevant conflicts of interest to declare.

The effect of sclareol on growth and cell cycle progression of human leukemic cell lines

1999 ◽  
Vol 23 (3) ◽  
pp. 217-234 ◽  
Author(s):  
Kostas Dimas ◽  
Dimitrios Kokkinopoulos ◽  
Costas Demetzos ◽  
Basilios Vaos ◽  
Marios Marselos ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Leukemic Cell ◽  
Cycle Progression ◽  
Human Leukemic Cell ◽  
Leukemic Cell Lines

scholarly journals Valproic acid inhibit non-small-cell lung cancer (A549 cell line) metastasis via inhibition of CD44v6 and Nm23H1

2019 ◽  
Author(s):  
Sorush Niknamian
Keyword(s):  
Lung Cancer ◽  
Valproic Acid ◽  
Cell Line ◽  
Histone Deacetylase ◽  
A549 Cell ◽  
Histone Deacetylase Inhibitors ◽  
Caspase 3 ◽  
Cancer Cell Line ◽  
Lung Cancer Cell Line ◽  
A549 Cell Line

Currently, epigenetic changes are considered to be one of the most important factors in the incidence of cancer and its metastasis to various tissues. It has been shown that CD44v6 and Nm23-H1 genes play a crucial role in the metastasis of various cancers. However, no study has been done on the effect of epigenetic factors on the expression of CD44v6 and Nm23-H1 genes in the lung cancer cell line, A549.Therefore, the present study investigated the role of a histone deacetylase inhibitor, valproic acid, on the expression of CD44v6 and Nm23-H1 genes and proteins in the A549 cell line. In this study, the A549 cell line was treated with valproic acid at concentrations of 2.2 mM, 4.5 mM, and 9 mM. We then investigated the expression of CD44v6 and Nm23-H1 genes and proteins, as well as the expression of MMP-2 and MMP-9 genes and caspase-3 activity. The results showed that valproic acid significantly decreased the expression of CD44v6 gene and protein and MMP-2 and MMP-9 genes, but increased the expression of Nm23H1 gene and protein and the activity of caspase-3 (p˂ 0.05). Our results showed that histone deacetylases affected the expression of CD44v6, Nm23-H1, MMP-2, and MMP-9, which are involved in metastasis. Therefore, the use of histone deacetylase inhibitors can be effective in the suppression of metastases and the treatment of lung cancer.

Charcterization of a Newly Established Megakaryo/Erythroid Leukemia Cell Line, JAS-R.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4327-4327
Author(s):  
Hisashi Yamada ◽  
Junko Horiguchi-Yamada ◽  
Tetsuaki Sekikawa
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Neutralizing Antibody ◽  
Chromosome Analysis ◽  
Erythropoietin Receptor ◽  
Leukemia Cell Line ◽  
Erythroid Cell ◽  
Leukemic Cell Lines

Abstract A few leukemic cell lines which express megakaryo/erythroid markers are available. We recently established a new cell line, designated JAS-R, from a 64-year-old patient with acute megakaryocytic leukemia (AML M7). Immunophenotyping showed that JAS-R cells were positive for CD4, CD7, CD13, CD33, CD41, CD61 and glycophorin A. Chromosome analysis was composite karyotype, but no major translocation abnormalities were observed. Electron-microscope examination disclosed that JAS-R had bleb like surface margin and a-granules in cytoplasm. Major four proteins which exist in a-granule were expressed high levels in JAS-R by RT-PCR. To further characterize JAS-R from four other megakaryo/erythroid cell lines (MEG-01, CMK, K562 and KU812), the comparison of gene expression profiling was studied by using oligo-DNA array. JAS-R was categorized as most different cell line among them. Of note, JAS-R secreted erythropoietin and expressed erythropoietin-receptor. But erythropoietin-neutralizing antibody failed to inhibit the growth of JAS-R cells. JAS-R may be useful for the further understanding of megakaryo and erythroid regulation and for the study of megakaryo/erythroid leukemogenesis.

A Histone Deacetylase Inhibitor Induces Apoptosis, Differentiation and Cell Cycle Changes in STI571 Resistant CML Cell Line with No Change in BCR-ABL Expression

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4235-4235
Author(s):  
Matityahu Shaklai ◽  
Yael Zimra ◽  
Inessa Belyaeva ◽  
Adina Aviram ◽  
Esther Rabizadeh
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Regulatory Protein ◽  
Resistance Mechanism ◽  
Chimeric Protein ◽  
Organic Cation Transporter ◽  
Resistant Cell Line ◽  
Myelogenous Leukemia

Abstract The hallmark of Chronic-myelogenous leukemia (CML) is high tyrosine kinase (TK) activity of the chimeric protein BCR-ABL, known to contribute to cell tumorogenity, resistance to apoptosis and differentiation. The gold-standard therapy for CML is treatment with the TK inhibitor Imatinib (STI571), but in some cases STI571 resistance appears due to BCR-ABL over expression, amplification or mutation in the ABL kinase domain. However, STI571 resistance due to other mechanisms which are BCR-ABL independent may not be excluded. BCR-ABL-positive cells can evade the inhibitory effect of STI571 by different mechanisms, such as reduced intake mediated by OCT-1 organic cation transporter-1), or enhanced efflux by Pgp, and, possibly, acquisition of compensatory mutations in genes other than BCR-ABL. One possible approach to overcome STI571 resistance involves the combination of Histone Deacetylase Inhibitors (HDACI). Previously we have shown: induced apoptosis, differentiation and reduced BCR-ABL protein levels in CML cell line (K562) by Pivaloyloxymethyl butyrate (Pivanex), a HDACI. In order to study STI571 resistance and the influence of HDACI on STI571 resistant CML cells we developed an STI571 K562 resistant cell line (K562-R). Our results show that s these K562-R cells had an IC50 33 times greater than that of K562 wild type (K562-S) cells. But K562-R cells had similar BCR-ABL protein and transcript levels as the K562-S and no mutations were found in the catalytic TK domain of K562-R cells. MDR protein expression and activity in K562-R were very low and similar to those of K562-S. However, the K562-R cell line had higher spontaneous apoptosis and G2M arrest and a slight but significant lower S phase. The K562-R cell line expressed more glycophorin A although spontaneous hemoglobin synthesis in K562-R cells was lower. Study of the signaling transduction pathway have shown no differences in p70S6, STAT3, Erk 1/2, CREB, Ikβ-a and JNK proteins levels (total and phosphorylated), however phosphorylated p38 was elevated in the K562-R cells. P21, a cell cycle regulatory protein, was higher in K562-R cells. HDACI induced viability loss, apoptosis and cell cycle changes in K562-R in a similar way to its effects on K562-S cells. However, it did not influence the levels of BCR-ABL. Taken together our results suggest that the STI571 resistance mechanism in these K562-R cells does not involve BCR-ABL but other regulatory mechanisms in the cell cycle, differentiation and signal transduction pathways. The higher spontaneous G2M arrest, apoptosis as well as the variable differentiation could be related to changes in p21 and p38 signaling pathways. These K562-R cells with no change in BCR-ABL may suggest a model for STI571 resistance that does not involve BCR-ABL directly. This data suggest, the possible benefit of HDACI treatment in CML STI571 resistant patient with no mutation or changes in BCR-ABL protein.

Dasatinib Is An Effective Inhibitor of Proliferation and Inducer of Apoptosis in the KASUMI Cell Line Bearing the T(8;21)(q22;q22) and the N822K KIT Mutation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5054-5054
Author(s):  
Vassiliki Pappa ◽  
F. Kontsioti ◽  
E. Liakata ◽  
S Papageorgiou ◽  
A. Spathis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cell Lines ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Kit Mutation ◽  
Free Survival ◽  
Leukemic Cell Lines ◽  
Imatinib Resistant ◽  
Proliferation And Apoptosis

Abstract Introduction. Within the group of core binding factor (CBF) AML, the presence of the t(8;21)(q22;q22) confers a favorable prognosis based on high complete remission rates and high survival probabilities. However within this subgroup the presence of KIT mutations and in some studies specifically mutations at codon 816 in exon 17 have been associated with inferior event free survival, relapse free survival, cumulative incidence of relapse and overall survival. Dasatinib a dual SRC/ABL kinase inhibitor is an active agent already approved for the treatment of imatinib resistant or intolerant chronic myelogenous leukemia which has shown in vitro activity against KIT exon 17 mutations including the D816 imatinib resistant mutation. The aim of the present study was the investigation of the activity of dasatinib on cell proliferation and apoptosis of leukemic cell lines with or without KIT mutations. Materials and methods. The leukemic cell lines ME-1, NB4 and KASUMI were cultured in RPMI. Following RNA extraction RT-PCR was performed for the amplification of the extracellular (exon 8,9), transmembrane/juxtamembrane (exon 10,11) and tyrosine kinase 2 domains (exon 17,18) of c-Kit.Following sequencing only the KASUMI cell line derived from a t(8;21)(q22;q22) AML was found to bear the N822K KIT mutation at exon 17, also described in patients’samples. The KASUMI, the K562 cell line bearing the t(9;22) used as a positive control and the NB4 cell line without KIT mutations used as a negative control, were subsequently cultured under the presence of dasatinib at the concentrations of 1nM, 10nM, 100nM, 500 nM. Cell proliferation, was determined at 24, 48, 72 h using the Cell Proliferation Elisa, BrDU protocol and apoptosis was determined by the method of annexin using flow cytometry at the same time points. Results The BrDU value of K562 cells at 48h without the drug was 1.046 significantly higher compared to those of cells cultured under the presence of Dasatinib at 1nM, 10nM, 100nM, 500 nM (0.6485, 0,5647, 0,4770, 0.4755 respectively) (p<0.001). Similarly the BrDU value of K562 cells without the drug at 72h was 1.320 significantly higher to those under the presence of the drug at 10, 100, 500 nM (0.8137, 0.7292, 0.6637 respectively) (p<0.001). The level of apoptosis was significantly induced by the drug at all concentrations at 24h(p<0.001) and at the concentrations of 10nM, 100nM, 500 nM at 48h (p<0.001) but not at 72h.Ôhere was no effect of the drug on the proliferation and apoptosis of the NB4 cell line. In the KASUMI cells there was a significant reduction of the BrDU values by the presence of dasatinib at the concentrations of 10nM, 100nM, 500nM at 48h (0.9517 vs 0.6462, 0.5653, 0.3467, p=0.038, 0.011, 0.002 respectively). The same was true at the concentrations of 100nM and 500nM at 72h (0.9538 vs 0.2412, 0.1907, p=0.002, 0.004 respectively). Dasatinib significantly increased the level of apoptosis of the KASUMI cells at 24h at 1nM, 10nM, 100nM (2.45 vs 1.41, 1.71, 2.18, p<0.001, <0.001, 0.026 respectively) At 48h dasatinib significantly increased the level of apoptosis at the concentrations of 1nM, 10nM, 100nM (0.84vs 1.03, 1.49, 2.81, p=0.02, p<0.001, p<0.001 respectively). At 72h there was a significant induction of apoptosis by the drug at all concentrations (0.16 vs 1.11, 1.94, 2.93, 1.88 p<0.001) Conclusion. Dasatinib is an effective suppressor of proliferation and inducer of apoptosis of the KASUMI cell line with the t(8;21)(q22;q22) and the N822K KIT mutation. These encouraging results need to be confirmed on patients’ cells with the view to integrate the drug in conventional chemotherapy regimens in future clinical trials.

Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Induces Apoptosis In Leukemic Cells through Mitochondria-Dependent Pathway

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2889-2889
Author(s):  
Mohammad Reza Mirlashari ◽  
Ingrid Randen ◽  
Jens Kjeldsen-Kragh
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Cell Types ◽  
Leukemic Cells ◽  
Caspase 8 ◽  
Apoptotic Pathway ◽  
Leukemic Cell Lines ◽  
Cell Death Pathway ◽  
Gsk 3Β

Abstract Abstract 2889 GSK-3β is a multifunctional kinase that plays a role in several signaling pathways. Due to the contradictory roles of GSK-3β as a mediator of both cell survival and apoptosis, we have examined the role of GSK-3β for proliferation and apoptosis in leukemic cell lines KG1a, K562 and CMK. GSK-3β was selectively inhibited by the small-molecule SB-415286. Treatment of leukemia cells with SB-415286 (40 μM) for 72 hr approximately halved cell growth in all three cell lines. SB-415286 also showed a concentration-dependent stabilization of intracellular β-catenin: In KG1a cells the mean fluorescence intensity (MFI) [± 95% CI] was 3.1 [± 1.7] in untreated cells vs. 423 [± 24] in treated cell. The figures for the K562 and CMK cell lines were: 2.8 [± 1.6] vs. 353.2 [± 11.1], and 6.8 [± 4.0] vs. 320.2 [± 23.7], respectively. Cell cycle analysis was carried out to examine if the growth inhibition was caused by arrest in cell cycle and/or induction of apoptosis. We found that SB-415286 caused cell cycle arrest in the G2/M phase and accumulation of events corresponding to the subG1 phase, indicative of DNA fragmentation. The subG1 population was 45%, 34% and 17% in KG1a, K562 and CMK cells, respectively. To confirm that the increase of the subG1 fraction represented an apoptotic effect of the GSK-3β inhibition, we analyzed phosphatidylserine (PS) externalization and plasma membrane integrity. We found that SB-415286 caused a considerable increase of the proportion of early apoptotic cells, i.e. cells that were annexin V-positive and 7-AAD-negative: Mean [± 95% CI] in KG1a cells increased from 6.2% [± 1.2%] in untreated cells to 38% [± 3.1%] in treated cells. The figures for the K562 and CMK cell lines were: 3.0% [± 1.2%] vs. 29% [± 3.3%], and 3.9% [± 1.0%] vs. 16.0% [± 1.1%], respectively. Apoptosis signaling can be initiated by extracellular (death receptor) and/or intracellular (mitochondrial) signals. Flow cytometric analysis of cells stained by a dual-fluorescent mitochondrial dye JC-1 showed that 5–11% of untreated leukemic cells had low mitochondrial membrane potential. After 72 hr exposure to SB-415286 the mean [±95% CI] loss of the mitochondrial potential was found in 23% [± 2.0%], 33% [± 3.5%] and 42% [± 3.8%], in CMK, K562 and KG1a cells, respectively. Since drug treatment in some cell types may result in activation of both the intrinsic or extrinsic cell-death pathway in a parallel manner, we investigated if the external pathway is involved in SB-415286-induced apoptosis. For this purpose we assessed caspase-8 activation by flow cytometry. After 72 hr of treatment of CMK, K562 and KG1a cells the caspase-8 activities compared, to untreated cells, had increased 3.7-fold, 3.9-fold, and 4.4-fold, respectively. In some cell types, the extrinsic cell-death pathway leads to the cleavage of Bid (pro-apoptotic member of the Bcl-2 family) by caspase-8, generating a truncated version of the protein (tBid) which in turn activates the mitochondrial apoptotic pathway. Therefore, we determined whether depolarization of the mitochondrial membrane in the leukemic cell lines was an effect of activated caspase-8 or a direct effect of SB-415286. For this purpose Z-IETD-FMK (25 μM), a specific inhibitor of caspase-8, was applied to the cells for 2 hr. We found that inhibition of caspase-8 did not prevent SB-415286-induced apoptosis assessed by PS externalization. This indicates that activation of caspase-8 is part of the intrinsic apoptotic pathway and occurs downstream of mitochondria membrane potential depolarization mediated by other caspases. Taken together, our observations suggest that inhibition of GSK-3β induces apoptosis of leukemic cells by depolarizing the mitochondria membrane. Thus, inhibition of GSK-3β could be an attractive target for treatment of leukemia. Disclosures: No relevant conflicts of interest to declare.

Chloride transport properties of human leukemic cell lines K562 and HL60

1984 ◽  
Vol 247 (1) ◽  
pp. C53-C60 ◽  
Author(s):  
S. Dissing ◽  
R. Hoffman ◽  
M. J. Murnane ◽  
J. F. Hoffman
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Leukemic Cell ◽  
High Energy ◽  
Energy Of Activation ◽  
Disulfonic Acid ◽  
Hl60 Cell ◽  
Human Leukemic Cell ◽  
Cl Transport ◽  
Leukemic Cell Lines

The Cl- transport characteristics of the human leukemic cell lines K562 and HL60, with erythroid and granulocytic phenotypic features, respectively, were investigated. Cl- effluxes were measured with 36Cl- under equilibrium conditions in both cell lines and were found to be three orders of magnitude slower than the unidirectional efflux of Cl- in normal erythrocytes. Induction of differentiation of the K562 cell line with hemin does not affect the rate of Cl- transport, while induction of the HL60 cell line with dimethyl sulfoxide results in a small decrease in the rate of Cl- transport. Cl- transport in both cell lines could be divided into two components. One component is inhibited by treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), displays counter-transport characteristics, and has a high energy of activation--all properties characteristic of the human erythrocyte-facilitated anion exchange system. The second component is insensitive to DIDS, is partially inhibited by furosemide, and has a low energy of activation.

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