Myeloid Leukemic Cell Lines HL-60 Research Articles

Histone Deacetylase Inhibitor Depsipeptide (FK228) Induces Apoptosis in Leukemic Cells by Facilitating Mitochondrial Translocation of Bax, Which Is Enhanced by Proteasome Inhibitor Bortezomib.

Histone deacetylase (HDAC) inhibitors are a promising candidate for molecular-targeted therapy for leukemia. In this study, we investigated the mechanisms of cytotoxic effects of depsipeptide (FK228), one of the most effective HDAC inhibitors against leukemia, using human myeloid leukemic cell lines HL-60 and K562. We found that FK228 activated caspase-9 and a subsequent caspase cascade by perturbing the mitochondrial membrane to release cytochrome c, which was almost completely blocked by overexpression of Bcl-2. The mitochondrial damage was caused by the translocation of Bax but not other proapoptotic Bcl-2 family proteins to the mitochondria. FK228 did not affect the interaction between Bax and Bax-adaptor proteins such as 14–3–3 theta and Ku70. FK228-induced apoptosis and mitochondrial translocation of Bax were markedly enhanced by the proteasome inhibitor bortezomib. The synergistic action of FK228 and Bortezomib was at least partly mediated through conformational changes of Bax, which facilitate its translocation to the mitochondria. These results suggest that the combination of HDAC inhibitors and proteasome inhibitors is useful for the treatment of leukemia especially in the context of molecular-targeted therapy. The status of Bcl-2 and Bax may influence the sensitivity of tumors to this combination, and thus, can be a target of further therapeutic intervention.

Whole-genome fingerprint of the DNA methylome during chemically induced differentiation of the human AML cell line HL-60/S4.

ABSTRACTEpigenomic regulation plays a vital role in cell differentiation. The leukemic HL-60/S4 [human myeloid leukemic cell line HL-60/S4 (ATCC CRL-3306)] promyelocytic cell can be easily differentiated from its undifferentiated promyelocyte state into neutrophil- and macrophage-like cell states. In this study, we present the underlying genome and epigenome architecture of HL-60/S4 through its differentiation. We performed whole-genome bisulphite sequencing of HL-60/S4 cells and their differentiated counterparts. With the support of karyotyping, we show that HL-60/S4 maintains a stable genome throughout differentiation. Analysis of differential Cytosine-phosphate-Guanine dinucleotide methylation reveals that most methylation changes occur in the macrophage-like state. Differential methylation of promoters was associated with immune-related terms. Key immune genes, CEBPA, GFI1, MAFB and GATA1 showed differential expression and methylation. However, we observed the strongest enrichment of methylation changes in enhancers and CTCF binding sites, implying that methylation plays a major role in large-scale transcriptional reprogramming and chromatin reorganisation during differentiation. Correlation of differential expression and distal methylation with support from chromatin capture experiments allowed us to identify putative proximal and long-range enhancers for a number of immune cell differentiation genes, including CEBPA and CCNF. Integrating expression data, we present a model of HL-60/S4 differentiation in relation to the wider scope of myeloid differentiation.

CD45 regulates homing and engraftment of immature normal and leukemic human cells in transplanted immunodeficient mice

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors invitro and their recovery in transplanted recipients invivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.

Histone Deacetylase Inhibitor Depsipeptide (FK228) Induces Apoptosis in Leukemic Cells by Facilitating Mitochondrial Translocation of Bax, Which Is Enhanced by the Proteasome Inhibitor Bortezomib

Histone deacetylase (HDAC) inhibitors are promising candidates for molecular-targeted therapy for leukemia. In this study, we investigated the mechanisms of cytotoxic effects of depsipeptide (FK228), one of the most effective HDAC inhibitors against leukemia, using human myeloid leukemic cell lines HL-60 and K562. We found that FK228 activated caspase-9 and a subsequent caspase cascade by perturbing the mitochondrial membrane to release cytochrome c, which was almost completely blocked by overexpression of Bcl-2. The mitochondrial damage was caused by the translocation of Bax but not other pro-apoptotic Bcl-2 family proteins to the mitochondria. FK228 did not affect the interaction between Bax and Bax adaptor proteins such as 14-3-3Θ and Ku70. FK228-induced apoptosis and mitochondrial translocation of Bax were markedly enhanced by the proteasome inhibitor bortezomib. The synergistic action of FK228 and bortezomib was at least partly mediated through conformational changes in Bax, which facilitate its translocation to the mitochondria. These results suggest that the combination of HDAC inhibitors and proteasome inhibitors is useful in the treatment of leukemia especially in the context of molecular-targeted therapy. The status of Bcl-2 and Bax may influence the sensitivity of tumors to this combination and thus can be a target of further therapeutic intervention.

Effect of Dioxin on Normal Human Hematopoietic and Leukemic Cells.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the prototypical agonist of the aryl hydrocarbon receptor (AhR), a member of the erb-A family that also includes receptors for steroids, thyroid hormones, peroxisome proliferators and retinoids. When bound to dioxin, the AhR can bind to DNA and alter the expression of various genes, including cytokines and growth factors. TCDD has a large number of biological effects, such as long-lasting skin disease, cardiovascular disease, diabete and cancer. An increase risk of Hodgkin's disease, non-Hodgkin's lymphoma and myeloid leukemia was observed after 15 years in the population exposed to dioxin following the 1976 accident in Seveso, Italy. In this study, we analysed the effect of escalating doses of TCDD on the human myeloid leukemic cell lines HL60 (promyelocytic leukemia) and K562 (chronic myelogenous leukemia), as well as on human CD34+ progenitor cells from the leukapheresis of normal donors stimulated with G-CSF. The possible specific modulation of gene expression induced by TCDD was then tested by means of macroarray analyses (Clontech), the results of which were successfully validated by real-time quantitative PCR on five candidate genes. The dioxin dose capable of inhibiting the growth of 50% of the colonies in semisolid medium (IC50 calculated using the CalcuSyn computer program) was 33 nM for HL60, 28 nM for K562, and 20 nM for the CD34+ cells. The HL60, K562 and CD34+ cells tested before and after exposure to 20 nM of TCDD allowed us to identify a series of modulated genes. The up-regulated genes included LUCA15 putative tumour suppressor, cyclin-D binding Myb-like protein (hDMP1), MAPK/ERK kinase 6, ras-related protein RAB2, RalB GTP-binding protein, transcription factor ZFM1, and BRCA1-associated ring domain protein (BARD1). The down-regulated genes included found c-myc, TNF alpha precursor, ICAM1, MIP1-beta, interferon gamma-induced protein precursor (gamma-IP10), granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemotactic protein 1 precursor (MCP1). All of these genes are variously involved in the processes of proliferation, differentiation and transformation in hematological and tumoral cell models. On the basis of these data, we can hypothesise that the inhibition of clonogenic potential and the gene expression induced by TCDD exposure on normal CD34+ progenitor cells plays a role in the neoplastic transformation of hemopoietic stem cells. This supports the epidemiological data indicating an increased hematological cancer risk in the population accidentally exposed to TCDD.

Effects of curcumin on proliferation and apoptosis in acute myeloid leukemia cells HL-60

To investigate the curcumin killing leukemia cellsin vitro,. Methods: The myeloid leukemic cell line HL-60 was studied by using cell culture, flow cytometrydetermining DNA content and TUNEL method measuring apoptotic cell percentage. Results: The data showed that curcumin selectively inhibited proliferation of acute myeloid leukemia (AML) HL-60 cell lines in a dose- and time-dependent manner. The growth inhibition rate was gradually increased and reached the peak at concentration of 25 (µmol/L curcumin at 24h. The sub-G, peak appeared after 12h treatment and was increased to 34.4% at 24h. The TUNEL method further certified that apoptotic cells reached 41% at the same phase. Conclusion: curcumin possesses obvious potent of anti-leukemia cell proliferation, which is contributed to the induction of HL-60 cells apoptosis. The concentration and action time of curcuminin vitro provide some reference for clinical use.

The influence of curcumin on the cell cycle of HL-60 cells and contrast study.

The mechanisms of curcumin inhibiting the proliferation of HL-60 cells were investigated. Acute myeloid leukemic cell line HL-60 was studied by using cell culture, NBT reduction, SABC method measuring BrdU incorporation rate, FCM measuring DNA contents and TUNEL method determining apoptotic cell percentage. Curcumin inhibited proliferation of HL-60 cells in a dose- and time-dependent manner. When the HL-60 cells were treated with 25 mumol/L curcumin for 48 h, the inhibitory rate was 60.71% +/- 1.20%. The study on BrdU incorporation rate and the distribution of DNA content and NBT reduction indicated that curcumin arrested the cells in G2/M phase of cell cycle at first, then in G0/G1 phase, the whole cell cycle progression was slowed down and DNA synthesis activities was halted. The arrested cells went to apoptosis instead of differentiation. The comparative study indicated that the ability of curcumin regulating the cell cycle of HL-60 cells was stronger than As2O3 and the acting points of curcumin were not completely consistent with those of VP-16. It was suggested that curcumin was able to regulate, to some extent, the G1/S and G2/M transmit checkpoints and disturb the HL-60 cell cycle to induce apoptosis.

Effects of concurrent use of rh-IFN-gamma and curcumin on the anti-proliferative capacity of HL-60 cells.

To understand the effect of rh-IFN-gamma on the ability of curcumin to kill HL-60 cells in vitro, the myeloid leukemic cell line HL-60 was studied by using cell culture. BrdU incorporation rate was examined by SABC, DNA content was determined by flow cytometry and apoptotic cell percentage was determined by TUNEL method. The results showed that curcumin inhibited proliferation of leukemic cells in a dose-dependent manner. When HL-60 cells were treated with 25 mumol curcumin for 24 h, the proliferative inhibitory rate was 43.75 +/- 2.00%. This effect could be enhanced obviously by IFN-gamma, the combined proliferative inhibitory rate increased to over 80%. The 5-BrdU incorporation rate and the distribution of DNA content indicated that curcumin could arrest cells in the G1/G0 and G2/M phase of cell cycle. At the same time, the sub-G1 peak (apoptotic peak) appeared. After IFN-gamma combined with curcumin, DNA synthesis rate decreased further. It showed a significant difference when compared with single drug group (P < 0.05). Meanwhile, sub-G1 peak also increased. The percentage of TUNEL positive cells was aslo increased. It is concluded that IFN-gamma can enhance the antiproliferative ability of curcumin against HL-60 cells.

Effect of concurrent use of rh-IL-3 and rh-GM-CSF on apoptosis of HL-60 cells induced by Ara-C.

The myeloid leukemic cell line HL-60 was studied by using DNA gel electrophoresis, flow cytomery, McAb C-myc, McAb Bcl-2 and CFU-L. From zero to 36 h, the apoptosis rates of 8 different phases and other indexes were observed. The results showed that with the prolonged time of drug incubation, apoptosis of HL-60 cells increased progressively. This effect can be enhanced obviously by rh-IL-3 and rh-GM-CSF. At the same time, the killed rate of leukemic cells by Ara-C induction was increased. C-myc expression was decreased and Bcl-2 expression did not display apparent change. Interestingly, the normal hemopoietic cells were not affected by these two kinds of cytokine. The theoretical basis was provided for concurrent use of rh-IL-3, rh-GM-CSF and cytotoxic drugs whose purpose is to elevate remission rate during the phase of induced remission of leukemia.

Bone marrow stromal cell blockade of human leukemic cell differentiation

Bone marrow (BM) stromal cell inhibition of leukemic cell differentiation was studied in cellular coculture experiments. In coculture, a significant percentage of cells from the human myeloid leukemic cell lines HL-60, PLB-985, and K562 adhere to fibroblastic KM- 102 BM stromal cells. A sensitive two-color immunofluorescence assay was developed to monitor stromal cellular effects upon leukemic cell differentiation. After chemical induction with 1 alpha,25- dihydroxyvitamin D3, strongly adherent HL-60 and PLB-985 cells were inhibited from differentiating into more mature monocytic cells, as measured by the monocytic surface marker CD14. In contrast, loosely adherent and nonadherent HL-60 and PLB-985 leukemic cells in the same cocultures, as well as both adherent and nonadherent K562 cells induced with phorbol ester, were not blocked in their capacity to differentiate. Scanning electron microscopy and intercellular dye transfer experiments correlated intimate stromal cell/leukemic cell interaction and intercellular communication with the blockade of leukemic cell differentiation. These studies indicate that there is significant variability among leukemic lines with respect to the nature of their adhesion to stromal cells. Moreover, the data implicate gap- junction formation as a potentially significant event in stromal cell- mediated leukemic cell regulation.

Bone Marrow Stromal Cell Blockade of Human Leukemic Cell Differentiation

AbstractBone marrow (BM) stromal cell inhibition of leukemic cell differentiation was studied in cellular coculture experiments. In coculture, a significant percentage of cells from the human myeloid leukemic cell lines HL-60, PLB-985, and K562 adhere to fibroblastic KM-102 BM stromal cells. A sensitive two-color immunofluorescence assay was developed to monitor stromal cellular effects upon leukemic cell differentiation. After chemical induction with 1α,25-dihydroxyvitamin D3, strongly adherent HL-60 and PLB-985 cells were inhibited from differentiating into more mature monocytic cells, as measured by the monocytic surface marker CD14. In contrast, loosely adherent and nonadherent HL-60 and PLB-985 leukemic cells in the same cocurtures, as well as both adherent and nonadherent K562 cells induced with phorbol ester, were not blocked in their capacity to differentiate. Scanning electron microscopy and intercellular dye transfer experiments correlated intimate stromal cell/leukemic cell interaction and intercellular communication with the blockade of leukemic cell differentiation. These studies indicate that there is significant variability among leukemic lines with respect to the nature of their adhesion to stromal cells. Moreover, the data implicate gap-junction formation as a potentially significant event in stromal cell-mediated leukemic cell regulation.

Treatment of myeloid leukemic cells with the phosphatase inhibitor okadaic acid induces cell cycle arrest at either G1/S or G2/M depending on dose.

The phosphatase inhibitor okadaic acid was found to induce cell cycle arrest of human myeloid leukemic cell lines HL-60 and U937 in a concentration- and time-dependent manner. Exposure to low concentrations of okadaic acid (2-8nM) for 24-48 hr caused greater than 70% of cells to arrest at G2/M, with up to 40% of the cells arrested in early mitosis. Cell viability decreased rapidly after 48 hr of treatment, and morphological and DNA structure analysis indicated that this was primarily due to the induction of apoptosis. The cells arrested in mitosis by 8 nM okadaic acid could be highly enriched by density gradient centrifugation and underwent apoptosis when further cultured either with or without okadaic acid, indicating that the effects of okadaic acid were irreversible. In contrast to the effects of low concentrations of okadaic acid, high concentrations (500 nM), inhibited proliferation in less than 3 hr. Remarkably, the majority of cells also entered a mitosis-like state characterized by dissolution of the nuclear membrane and condensation and partial separation of chromosomes. However, these cells had a diploid content of DNA, indicating that the cell cycle arrest occurred at G1/S with premature chromosome condensation (PCC), rather than at G2/M. If cells were first blocked at G1/S with hydroxyurea and then treated with okadaic acid, greater than 90% developed PCC in less than 3 hr without replicating their DNA. Caffeine was not able to induce PCC in these cells, either with or without prior inhibition of DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical characterization of a 183 KD myeloid-specific-DNA- binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1

A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.

Immunohistochemical Characterization of a 183 KD Myeloid-Specific–DNA-Binding Protein in B5 Fixed, Paraffin-Embedded Tissues, and Bone Marrow Aspirates by Monoclonal Antibody BM-1

AbstractA monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-0 showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.

Isolation and Characterization of a T Lymphocyte-Derived Differentiation Inducing Factor for the Myeloid Leukemic Cell Line HL-60

Mitogen-stimulated mononuclear blood cells produce differentiation inducing factors (DIFs) for the promyelocytic cell line HL-60. We report that DIF is produced constitutively by a malignant T lymphocyte line HUT-102. DIF was purified 7,000-fold from HUT-102 conditioned media by utilizing ion-exchange chromatography with DEAE-Sepharose, gel chromatography, Blue-Sepharose chromatography, and preparative SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The final preparation is susceptible to protease treatment, has a molecular weight of 46,000, as determined by SDS-PAGE and approximately 55,000 by gel filtration, has an isoelectric point of approximately 5.2, does not adhere to lectin-Sepharose and is resistant to periodate oxidation, and is free of colony-stimulating factor. DIF induced maturation of HL-60 into phagocytizing nitro blue tetrazolium reducing cells with the morphological characteristics of myelomonocytic or monocyte-like cells. An activity, co-chromatographing with DIF, acts synergistically with retinoic acid to induce maturation not only of HL-60, but also of the monoblast-like cell line U-937 (measured as percentage of cells reducing NBT).

Human granulocyte-specific nuclear antigen(s). II. Detection of antigens in human proliferative syndromes.

Antisera raised to dehistonized chromatin from isolated normal human granulocytes revealed the presence of chromatin-associated antigens specific for the human neutrophils that appear during late stages of myeloid cellular differentiation. Immunological specificity was demonstrated by C fixation, immunodiffusion, and immunocytochemical reactions. Chromatin prepared from both normal granulocytes and specimens of myeloid leukemia showed immunologic reactivity. Although the normal antigens were detected in a specimen of CML, the position of immunodiffusion precipitin lines was different from that obtained with normal granulocyte chromatin. In addition, chromatin prepared from the myeloid leukemic cell line HL-60 expressed only one of the three precipitin bands normally found in immunodiffusion. The immunocytochemical staining reaction was confined to the nucleus of mature neutrophils in normal peripheral blood smears. Greater than 90% of cells in peripheral blood specimens of CML showed positive immunocytochemical nuclear staining. In other types of leukaemia, the normal mature granulocyte reacted with antiserum, but the nonmyeloid leukemic cells in these specimens did not. The specificity of immunologic reactions described here suggests the usefulness of nuclear antigens as cell markers.