Differentiation Of Leukemia HL-60 Cells Research Articles

CDDO Induces Differentiation through PTEN Expression in HL-60 Cells

Acute promyelocytic leukemia (APL) represents a group of haematological malignancies characterized by blocked differentiation. Cell differentiation has been as a strong target of treatment of APL. Peroxisome proliferator-activated receptors gamma (PPAR-γ) play an important role in the differentiation of APL cell lines. Previous study has shown that PPAR-γ ligands induce neutrophil/ monocytic differentiation in human acute promyelocytic leukemia HL-60 cells. It has recently been shown that the novel synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation in leukemia HL-60 cells. In this study we established a CDDO-induced differentiation model for the treatment of APL. We evaluated for the surface antigen expression associated with myeloid maturation, CD11b and CD14 by flow cytometric analysis and RT-PCR. We demonstrated that CD11b and CD14 increased as CDDO -induced differentiation. In addition, CDDO induced a HL-60 differentiation through the regulation of PTEN expression. PTEN also has various cellular function such as cell differentiation. PPAR-γ could synergistically up-regulate PTEN in human leukemia cells and consequently stimulate the differentiation of these cells We confirmed that suppression of PTEN with the inhibitor (SF1760) caused reduction of differentiated HL-60 cells. Our results suggest PPAR-γ an important role in controlling the differentiation of leukemia cells via the regulation of PTEN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MAP kinase-dependent autophagy controls phorbol myristate acetate-induced macrophage differentiation of HL-60 leukemia cells

We investigated the mechanisms and the role of autophagy in the differentiation of HL-60 human acute myeloid leukemia cells induced by protein kinase C (PKC) activator phorbol myristate acetate (PMA). PMA-triggered differentiation of HL-60 cells into macrophage-like cells was confirmed by cell-cycle arrest accompanied by elevated expression of macrophage markers CD11b, CD13, CD14, CD45, EGR1, CSF1R, and IL-8. The induction of autophagy was demonstrated by the increase in intracellular acidification, accumulation/punctuation of autophagosome marker LC3-II, and the increase in autophagic flux. PMA also increased nuclear translocation of autophagy transcription factors TFEB, FOXO1, and FOXO3, as well as the expression of several autophagy-related (ATG) genes in HL-60 cells. PMA failed to activate autophagy inducer AMP-activated protein kinase (AMPK) and inhibit autophagy suppressor mechanistic target of rapamycin complex 1 (mTORC1). On the other hand, it readily stimulated the phosphorylation of mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) via a protein kinase C-dependent mechanism. Pharmacological or genetic inhibition of ERK or JNK suppressed PMA-triggered nuclear translocation of TFEB and FOXO1/3, ATG expression, dissociation of pro-autophagic beclin-1 from its inhibitor BCL2, autophagy induction, and differentiation of HL-60 cells into macrophage-like cells. Pharmacological or genetic inhibition of autophagy also blocked PMA-induced macrophage differentiation of HL-60 cells. Therefore, MAP kinases ERK and JNK control PMA-induced macrophage differentiation of HL-60 leukemia cells through AMPK/mTORC1-independent, TFEB/FOXO-mediated transcriptional and beclin-1-dependent post-translational activation of autophagy.

Trdmt1 3'-untranslated region functions as a competing endogenous RNA in leukemia HL-60 cell differentiation.

Severe blockage in myeloid differentiation is the hallmark of acute myeloid leukemia (AML). Trdmt1 plays an important role in hematopoiesis. However, little is known about the function of Trdmt1 in AML cell differentiation. In the present study, Trdmt1 was up-regulated and miR-181a was down-regulated significantly during human leukemia HL-60 cell differentiation after TAT-CT3 fusion protein treatment. Accordingly, miR-181a overexpression in HL-60 cells inhibited granulocytic maturation. In addition, our “rescue” assay demonstrated that Trdmt1 3′-untranslated region promoted myeloid differentiation of HL-60 cells by sequestering miR-181a and up-regulating C/EBPα (a critical factor for normal myelopoiesis) via its competing endogenous RNA (ceRNA) activity on miR-181a. These findings revealed an unrecognized role of Trdmt1 as a potential ceRNA for therapeutic targets in AML.

MicroRNA-16 Regulates Myeloblastosis Oncogene Expression to Affect Differentiation of Acute Leukemia Cells.

This study was designed to evaluate the effects of micro-RNA-16 (miR-16)-regulated expression of myeloblastosis oncogene (MYB) on the differentiation of acute leukemia cells, the expressions of miR-16 and MYB mRNA, and protein in differently differentiated leukemia cells were detected by real-time PCR and western blot. 1,25-Dihydroxyvitamin D3 (1,25 D3) induced monocytic differentiation of HL60 cells, and the resulting changes in miR-16 and MYB expressions were detected. Morphology of the cells induced by 1,25 D3, after being transfection with miR-16 mimics, was observed by Wright-Giemsa staining. The expression of mononuclear cell surface marker CD14 was detected by flow cytometry. Minimum miR-16 was expressed in early-differentiation KG-1a cells, while late-differentiation U937 and THP-1 cells had higher expressions (p < 0.01). The expressions of MYB changed oppositely. During the monocytic differentiation of HL60 cells, miR-16 expression showed a time-dependent increase, but MYB expression gradually decreased. Overexpression of miR-16 in HL60 cells promoted 1,25 D3-induced morphological changes and CD14 expression (p < 0.05). MR-16 facilitated the monocytic differentiation of leukemia HL60 cells by negatively regulating MYB expression.

Role of Sphingolipids and Metabolizing Enzymes in Hematological Malignancies.

Sphingolipids such as ceramide, sphingosine-1-phosphate and sphingomyelin have been emerging as bioactive lipids since ceramide was reported to play a role in human leukemia HL-60 cell differentiation and death. Recently, it is well-known that ceramide acts as an inducer of cell death, that sphingomyelin works as a regulator for microdomain function of the cell membrane, and that sphingosine-1-phosphate plays a role in cell survival/proliferation. The lipids are metabolized by the specific enzymes, and each metabolite could be again returned to the original form by the reverse action of the different enzyme or after a long journey of many metabolizing/synthesizing pathways. In addition, the metabolites may serve as reciprocal bio-modulators like the rheostat between ceramide and sphingosine-1-phosphate. Therefore, the change of lipid amount in the cells, the subcellular localization and the downstream signal in a specific subcellular organelle should be clarified to understand the pathobiological significance of sphingolipids when extracellular stimulation induces a diverse of cell functions such as cell death, proliferation and migration. In this review, we focus on how sphingolipids and their metabolizing enzymes cooperatively exert their function in proliferation, migration, autophagy and death of hematopoetic cells, and discuss the way developing a novel therapeutic device through the regulation of sphingolipids for effectively inhibiting cell proliferation and inducing cell death in hematological malignancies such as leukemia, malignant lymphoma and multiple myeloma.

Wnt signaling is involved in 6-benzylthioinosine-induced AML cell differentiation.

BackgroundWe previously demonstrated that 6-benzylthioinosine (6-BT) could induce the differentiation of a subset of acute myeloid leukemia (AML) cell lines and primary AML cells regardless of their cytogenetics. In this study we investigated whether Wnt signaling pathways played roles in 6-BT-induced differentiation of AML cells.MethodsWe induced differentiation of HL-60 leukemic cells and primary AML cells in vitro using 6-BT. Real-time PCR (qPCR), western blot, and luciferase assays were used to examine the molecules’ expression and biological activity in canonical and noncanonical Wnt signaling pathways. AML cell differentiation was measured by the Nitroblue tetrozolium (NBT) reduction assay.Results6-BT regulated the expression of both canonical and non-canonical Wnt signaling molecules in HL-60 cells. Both 6-BT and all-trans-retinoic-acid (ATRA) reduced canonical Wnt signaling and activated noncanonical Wnt/Ca2+ signaling in HL-60 cells. Pre-treatment of HL-60 cells with an inhibitor of glycogen synthase kinase-3β (GSK-3β), which activated canonical Wnt signaling, partly abolished the differentiation of HL-60 cells induced by 6-BT. Pre-treatment of HL-60 cells with an inhibitor of protein kinase C (PKC), resulting in inactivation of non-canonical Wnt/Ca2+ signaling, abolished 6-BT-induced differentiation of HL-60 cells. Several molecules in the non-canonical Wnt/Ca2+ pathway were detected in bone marrow samples from AML patients, and the expression of FZD4, FZD5, Wnt5a and RHOU were significantly reduced in newly diagnosed AML samples compared with normal controls.ConclusionsBoth canonical and non-canonical Wnt signaling were involved in 6-BT-induced differentiation of HL-60 cells, and played opposite roles in this process. Wnt signaling could be involved in the pathogenesis of AML not only by regulating self-renewal of hematopoietic stem cells, but also by playing a role in the differentiation of AML cells.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-886) contains supplementary material, which is available to authorized users.

Abstract 4192: Genome-wide microRNA and functional genomics analysis during differentiation of acute promyelocytic leukemia to granulocytes.

Abstract MicroRNA (miRNAs) are short noncoding RNAs that play important roles in human diseases, including cancer, and function to regulate gene expression of critical processes, such as apoptosis, cell proliferation and differentiation, by either translational inhibition or mRNA degradation. However, little is known about the role of miRNAs in granulopoiesis and acute myeloid leukemia (AML). We performed genome-wide miRNAs and functional genomics analysis in the acute promyelocytic leukemia (APL) human cell line HL-60 during its differentiation towards granulocytes, by using miRNA microarrays Exiqon platform, quantitative real time polymerase chain reaction (qRT-PCR) and Ingenuity Pathways Analysis(IPA). Among 1279 human miRNAs analyzed, we identified a unique signature of 86 differentially expressed miRNAs between control and DMSO-treated cells (at p&amp;lt;0.05), 51 of them were up-regulated and 35 of were down-regulated, which have not previously identified as regulators of hematopoietic differentiation. Our data showed that miRNA expression profiling revealed distinctive miRNA signatures that correlated with morphological changes in the cell and nucleus, granule formation, and chromatin condensation, all these processes being consistent with neutrophil maturation. Transmission electron microscopy (TEM) showed large mitochondria and revealed the presence of multiple autophagosome-like vacuoles with double-membrane structures in neutrophil-like differentiated cells. Specific miRNAs, such as miR-125a-5p and miR-483-3p, appeared to be associated with chromatin remodeling and nuclear protein expression, such as chromosome condensation (CHC1L) and histone family member X (H1FX), during HL-60 cell differentiation towards granulocytes. We found by gain-of-function experiments that miRNA-125a-5p and miR483-3p promoted granulocytic differentiation of HL-60 leukemic cells as confirmed by nitroblue tetrazolium (NBT) staining and mature myeloid cell marker expression, while miR-17-92 cluster attenuated granulocytic differentiation. Therefore miRNA-125a-5p, miRNA-483-3p up-regulation and miRNA-17-92 cluster down-regulation provide new insight into the mechanism of granulopoiesis. Molecular network analysis of miRNA targetome during APL differentiation showed a regulation by individual miRNAs that constitute a biological network of functionally-associated molecules in human cells, closely linked to pathological events involved in cancer, p53 signaling, gene expression, cellular growth and differentiation. These findings provide unique molecular insights into how APL malignant cells can be differentiated towards terminal neutrophil-like cells, which might lead to the identification of novel molecular targets in leukemia differentiation therapy. Citation Format: El Habib Dakir, Jun-ichi Satoh, Faustino Mollinedo. Genome-wide microRNA and functional genomics analysis during differentiation of acute promyelocytic leukemia to granulocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4192. doi:10.1158/1538-7445.AM2013-4192

PBK/TOPK Expression During TPA-Induced HL-60 Leukemic Cell Differentiation

This study concerns expression of PBK/TOPK during differentiation of HL-60 leukemic cells induced by tetradecanoyl phorbol acetate (TPA). Wright-Giemsa staining was performed to observe morphological changes in the HL-60 cells, and flow cytometry was used to assess the cell cycle and CD11b, CD14, CD13, and CD33 expression. PBK/TOPK levels were determined by Western blot analysis. After treating HL60 cells with 5.1×10⁻⁹ mmol/L of TPA for three days, the number of nitroblue-tetrazolium-positive cells and CD11b, CD13, and CD14 expression increased, whereas the PBK/TOPK levels decreased. TPA can inhibit proliferation and induce differentiation of HL60 cells of the granulocytic or monocytic lineage. PBK/TOPK expression was downregulated during this process, whereas the Pho-PBK/TOPK expression was increased.

Synthesis of C 6-epimer derivatives of diacetoxy acetal derivative of santonin and their inducing effects on HL-60 leukemia cell differentiation

Induction of differentiation is a new and promising approach to leukemia therapy, well illustrated by the treatment of acute promyelocytic leukemia with 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] or all-trans retinoic acid (ATRA). Using combination of either 1,25-(OH)(2)D(3) or ATRA and chemotherapy, adverse effects 1,25-(OH)(2)D(3) or ATRA such as hypercalcemic effects have decreased, and long-term survival has improved. In a previous study, we demonstrated that santonin could be chemically modified into a diacetoxy acetal derivative of santonin with strong differentiation-inducing activity. In this study, we further synthesized C(6)-epimer derivatives of diacetoxy acetal derivative of santonin and tested their effects on HL-60 cell differentiation. Some of the C(6)-epimer derivatives themselves induced increases in cell differentiation. Especially, (11S)-3,3-(ethylenedioxy) eudesmano-13-ol-6β-acetate (7) was demonstrated to induce differentiation with larger than 80% of the cells attaining a differentiated phenotype. Importantly, 7 strongly enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either low doses of 1,25-(OH)(2)D(3) or ATRA. The ability to enhance the differentiation potential of 1,25-(OH)(2)D(3) or ATRA by 7 may improve outcomes in the therapy of acute promyelocytic leukemia.

Gnidilatimonoein from Daphne mucronata induced differentiation and apoptosis in leukemia cell lines

Among various treatments, plants play a crucial role in cancer chemotherapy. Base on literature data, different species of the thymeleaeceae family have been found to be good sources for anticancer agents. Therefore, our laboratory has initiated a research project on evaluating the anticancer properties of the Iranian medical plant, Daphne mucronata (Thymeleaeceae). Gnidilatimonoein is a new diterpene ester compound that is purified from Daphne mucronata with antiproliferative and anti-metastatic effects in different tumor cell lines. Based on the inhibitory role of this agent on the cell cycle progression of K562 cell line, we were interested to check gnidilatimonoein for induction of differentiation and apoptosis in different human leukemia ML-1, HL-60 and K562 cells. Cell viability was determined by trypan blue exclusion method. Cell differentiation was studied in terms of morphology, NBT reduction in leukemia cells (HL-60 and ML-1) and benzidine staining in erythroleukemia (K562) cells. Apoptosis was detected by fluorescence microscopy (Acridine orange/Ethedium bromide double staining), annexineV flow cytometry and DNA electrophoresis. After 48 hours of treatment with 10 µM gnidilatimonoein, 80-70% of HL-60 and ML1 cells showed NBT reduction. Indeed gnidilatimonoein induced monocyte differentiation of myeloid leukemia HL-60 and ML-1 cells but not K562 erythroluekemia cells. In this condition, we observed the growth inhibitory effect (60%) without substantial loss of viability (

Chromatin remodeling: recruitment of histone demethylase RBP2 by Madl for transcriptional repression of a Myc target gene, telomerase reverse transcriptase

The Myc/Max/Mad network transcription factors are known to govern target gene expression through recruiting histone acetyltransferases or deacetylases. In the present study, we show that Mad1 recruits the histone demethylase RBP2 to the Myc target telomerase reverse transcriptase (hTERT) gene promoter to repress transcription. With differentiation of leukemic HL60 cells, Mad1 and RBP2 were both up-regulated, interacted, and cooccupied the hTERT promoter accompanied by histone H3-K4 demethylation. In immortalized p493-6 B cells, shutting down c-Myc led to the accumulation of Mad1 and RBP2 at hTERT promoter and diminished hTERT mRNA expression. When RBP2 was depleted, hTERT expression was significantly enhanced, coupled with dissociation of RBP2 with and increased H3-K4 methylation at the hTERT promoter in p493-6 cells. Moreover, RBP2 and Mad1 were present on the hTERT promoter in human fibroblasts having a silent hTERT gene, and RBP2 depletion resulted in gene derepression. Taken together, Mad1 recruits RBP2 to the hTERT promoter that, in turn, demethylates H3-K4, thereby contributing to a stable repression of the hTERT gene in normal or differentiated malignant cells. Our findings reveal a novel mechanism through which the Myc/Max/Mad network proteins control their target gene transcription and provide insights into mechanisms underlying telomerase silencing and activation.

Regulation of CD11b transcription by decreasing PRC2 and increased acH4 level during ATRA-induced HL-60 differentiation

Polycomb repressive complex 2 (PRC2), which mediates trimethylation of lysine 27 on histone H3 (K27me3), plays an important role in many types of stem cell differentiation. Here, we try to reveal how PRC2, PRC2-mediated repressive histone marker H3K27me3, and active histone marker histone H4 acetylation (acH4) regulate the CD11b transcription during alltrans retinoic acid (ATRA)-induced HL-60 leukemia cell differentiation. By using quantitative real-time polymerase chain reaction (qPCR) and western blot analysis, we found that the mRNA and protein expression levels of two members of PRC2 were decreased during ATRA-induced HL-60 differentiation, respectively. When treated with ATRA for 72 h, the EZH2 and SUZ12 mRNA levels were decreased to 35% and 38% of the control group, respectively. At the same time, the granulocytic mature surface marker CD11b expression was increased significantly at mRNA level detected by qPCR and protein level detected by flow cytometry. By using chromatin immunoprecipitation assay, we compared the local changes in SUZ12 binding and PRC2-mediated H3K27me3 at the promoter of CD11b during ATRA-induced HL-60 differentiation. Both the levels of SUZ12 binding and PRC2-mediated H3K27me3 at the promoter of CD11b were decreased for 4.1 and 3.8 folds, respectively. And we also found the increase in the acH4 level up to 4 folds after 72 h of ATRA treatment. These results suggested that the histone modification including PRC2-mediated repressive histone marker H3K27me3 and active histone marker acH4 may involve in CD11b transcription during HL-60 leukemia cells reprogramming to terminal differentiation.

Gene expression analysis of human promyelocytic leukemia HL-60 cell differentiation and cytotoxicity induced by natural and synthetic retinoids

Aims This study analyzed gene expression profiles of human promyelocytic leukemia HL-60 cells treated with natural and synthetic retinoids ( ATRA, RII and R9158), in an attempt to investigate the structure–function relationship of the retinoids in inducing cell differentiation and cytotoxicity. Main methods Flow cytometry was used to determine cell cycle changes in HL-60 cells following treatment (1.0 μM) with natural and synthetic retinoids ( ATRA, RII and R9158), and cDNA microarrays were used to monitor the gene expression profiles of HL-60 cells treated with the various retinoids. Key findings Consistent with retinoid-induced cell differentiation, treatment with these three retinoids correlated with an increase in the percentage of cells arrested in the G1/G0 phase of the cell cycle. Microarray analysis showed upregulation of known differentiation genes, adhesion molecules, and the oxidase activation pathway following retinoid treatment. Differential expression of several genes was observed in HL-60 cells treated with the three retinoids. For example, tissue remodeling protein genes, ubiquitin genes, and signal transduction genes were highly expressed in ATRA- and R9158-treated HL-60 cells, but remained unchanged in HL-60 cells treated with RII. Significance The above findings suggest that the differentiation of HL-60 cells induced by the three retinoids occurs through similar pathways, and that there exists a structure–function relationship regarding retinoids and the induction of cell differentiation and cytotoxicity.

Mir-29c and Mir-424 Are Novel Myeloid Differentiation-Associated MicroRNAs in Acute Promyelocytic Leukemia.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. Recent studies showed that they are critically involved in hematopoietic differentiation and function by a coordinating multi-target repression of hematopoiesis-related genes. To identify miRNAs involved in the pathogenesis of acute promyelocytic leukemia (APL), characterized by the t(15;17) translocation, we performed TaqMan Low Density Array-based miRNA expression profiling on blast cells from an APL patient under all-trans retinoic acid (ATRA) treatment. Although recent reports investigated miRNA expression patterns in APL blast cells and cell lines subjected to ATRA in vitro, to our knowledge this is the first study that relies on cells from an APL patient treated with ATRA in vivo. Since the downregulation of the PML-RARA transcript cannot be assessed within a time period of a few days, we monitored effective ATRA treatment by measuring mRNA downregulation of the panleukemic marker Wilms' tumor (WT)-1. WT1 mRNA levels decreased 64% and 92% at day 3 and 6 upon ATRAtherapy, respectively. Total RNA obtained at diagnosis and at days 3/6 following ATRA therapy were screened for expression patterns of 384 human miRNAs including two endogenous controls, RNU44 and RNU48, for normalization of miRNA expression. Since these controls were regulated upon ATRA treatment, we normalized miRNA expression to miR-93, which showed stable expression in our samples. Consistent with previous in vitro APL miRNA profiling data, the granulocyte-specific miR-223 was induced 6.6-fold at day 6 upon ATRA treatment. For further analysis, we focused on two hematopoietic lineage-specific miRNAs, miR-29c and miR-424 that have not yet been associated with neutrophil development. miR-29c and miR-424 were upregulated 6.5- and 6.0-fold at day 6 in response to ATRA, respectively. Induction of these miRNAs was confirmed by individual real-time RT-PCR assays. Moreover, expression of miR-29c and miR-424 was further investigated in NB4 and HT93 APL cell lines. In both cell lines, miR-424 was upregulated in response to ATRA similar to the patient samples, suggesting a role for miR-424 in granulocytic differentiation in addition to that described in macrophage development. miR-29c, however, showed an upregulation in HT93 but not in NB4 cells implying cell type specific regulation. Additionally, we tested the involvement of miR- 29c in macrophage differentiation of HL60 leukemic cells using phorbol 12-myristate 13-acetate (PMA) as a differentiating agent. Interestingly, miR-29c showed an 8.0- fold upregulation similar to an 8.7-fold induction of miR-424, a known target of the transcription factor PU.1 upon PMA treatment. Based on the similar regulation of miR-29c and miR-424 and the presence of several putative PU.1 binding elements in the miR-29c promoter, we are currently investigating whether miR-29c is a novel transcriptional target of PU.1. A confirmed target of miR-29c is the protein DNA methyltransferase (DNMT 3A and 3B), which is overexpressed in myeloid leukemias. Therefore, induction of miR-29c during myelopoiesis might be needed to target DNMT. In conclusion, we propose a novel association of miR-29c and miR-424 with ATRA-induced neutrophil differentiation.

The Novel Retinoid, 9cUAB30, Inhibits Telomerase and Induces Apoptosis in HL60 Cells

Telomerase, a ribonucleoprotein important to neoplastic immortality, is up-regulated in approximately 85% of cancers, including leukemias. In this study, 9cUAB30, a novel retinoic acid, resulted in differentiation of HL60 leukemia cells as indicated by morphologic changes characteristic of granulocytes. It also caused a down-regulation of hTERT gene expression and a decrease in telomerase activity. Telomerase inhibition was followed by loss of proliferative capacity, induction of apoptosis, and partial differentiation. These findings demonstrate the effectiveness of 9cUAB30 at inhibiting telomerase activity by down-regulating hTERT gene expression in human leukemic cells.

CIP2A Is Overexpressed in Gastric Cancer and Its Depletion Leads to Impaired Clonogenicity, Senescence, or Differentiation of Tumor Cells

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogenic factor stabilizing c-MYC protein and driving cellular transformation. We determine whether CIP2A expression can serve as marker for gastric cancer and investigate the mechanism underlying CIP2A-mediated transformation and cell proliferation. Normal and malignant gastric tissues derived from 37 patients with gastric cancer were analyzed for CIP2A expression using reverse transcription-PCR and immunohistochemical staining. Gastric and other cell lines with different p53 and pRB backgrounds were used to inhibit CIP2A expression using small interfering RNA and then examined for clonogenic potentials, senescence, or differentiation. CIP2A mRNA was present in 34 of 37 (90%) of tumor specimens but absent in 27 of 37 (73%) of matched normal gastric mucosa. In 10 adjacent normal tissues with detectable CIP2A mRNA, 6 of them exhibited much weaker levels of CIP2A compared with their corresponding tumors. Thus, a total of 32 (87%) gastric cancer samples overexpressed CIP2A. CIP2A protein expression was readily detectable in the tumor tissues but absent in normal gastric mucosa. Depleting CIP2A expression substantially inhibited growth and clonogenic capabilities of tumor cell lines independently of p53 and pRB pathways. Gastric cancer-derived AGS cells underwent senescence following the inhibition of CIP2A expression. Moreover, CIP2A depletion triggered partial differentiation of leukemic HL60 cells. CIP2A in tumor cells is required for sustained proliferation by preventing cell growth arrest, senescence, or differentiation and its expression is significantly (P < 0.001) discriminatory between normal and cancerous gastric tissue.

Interferon-α enhances artemisinin-induced differentiation of HL-60 leukemia cells via a PKCα/ERK pathway

Interferon-α (IFN-α) has been used in the treatment of several cancers, including chronic myeloid leukemia. Artemisinin, a sesquiterpene lactone endoperoxide that exists in several medicinal plants, is a well known anti-malarial agent. We previously reported that artemisinin by itself caused a relatively low level of HL-60 cell differentiation. In this study, we investigated the effects of IFN-α in combination with artemisinin on cell growth and differentiation in HL-60 leukemia cells. Combination of IFN-α and artemisinin synergistically induced the levels of leukemia cell differentiation, although IFN-α by itself did not affect cell proliferation and differentiation. The increased cell differentiation by IFN-α and artemisinin was significantly suppressed by the inhibitors for protein kinase C (PKC), extracellular signal-regulated kinase (ERK) and jun N-terminal kinase (JNK), but not by the inhibitors for phosphatidylinositol 3-kinase (PI3-K) and p38 mitogen-activated protein kinase (MAPK). Furthermore, co-treatment with IFN-α increased levels of PKCα and phosphorylated ERK. Taken together, these results indicate the enhancement of artemisinin-induced HL-60 cell differentiation by IFN-α through the activation of a PKCα/ERK signaling pathway, and suggest a possible use of IFN-α and artemisinin in the treatment of leukemic diseases.

Synthesis of DAAS derivatives and their enhancement of HL-60 leukemia cell differentiation

DAAS is the diacetoxy acetal derivative of a-santonin and induces HL-60 cell differentiation into granulocytes. In this report, we investigated the structure-activity relationship (SAR) of DAAS derivatives in the differentiation of human HL-60 leukemia cells. Although its derivatives themselves had less effect on HL-60 cell differentiation than DAAS, the monoacetyl derivative, 2, mainly induced HL-60 cell differentiation. Moreover, compound 2 synergistically enhanced all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation when combined with 50 nM ATRA, a well-known differentiation inducer. This enhancing effect is similar to that of DAAS in ATRA-induced differentiation.

Specific association of increased vascular endothelial growth factor expression and its receptors with macrophage differentiation of HL-60 leukemia cells

We investigated the gene expression profiles of vascular endothelial growth factor (VEGF) and its receptors in HL-60 leukemia cells. In the VEGF family, both mRNA and protein expression of VEGF-C were up-regulated in phorbol myristate acetate (PMA)-differentiated HL-60 cells. We detected two bands of ∼31 and ∼60kDa in cell lysates, and the higher expression of ∼31kDa band was further increased after stimulation with tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS). A ∼31kDa VEGF-C protein was also detected in conditioned media from PMA-differentiated HL-60 cells after LPS stimulation. The mRNA expression of VEGFR-1, VEGFR-2, and neuropilin-1 (NRP-1) was markedly up-regulated in PMA-differentiated HL-60 cells, corresponding to the results from VEGF binding studies, in which VEGF binding activity was increased in PMA-differentiated HL-60 cells. These did not occur in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. The expression of VEGF-C and VEGF receptors is regulated specifically in HL-60 cells during macrophage differentiation.

Lysine-Specific Demethylase 1 (LSD1) Is Required for the Transcriptional Repression of the Telomerase Reverse Transcriptase (hTERT) Gene

BackgroundLysine-specific demethylase 1 (LSD1), catalysing demethylation of mono- and di-methylated histone H3-K4 or K9, exhibits diverse transcriptional activities by mediating chromatin reconfiguration. The telomerase reverse transcriptase (hTERT) gene, encoding an essential component for telomerase activity that is involved in cellular immortalization and transformation, is silent in most normal human cells while activated in up to 90% of human cancers. It remains to be defined how exactly the transcriptional activation of the hTERT gene occurs during the oncogenic process.Methodology/Principal FindingsIn the present study, we determined the effect of LSD1 on hTERT transcription. In normal human fibroblasts with a tight hTERT repression, a pharmacological inhibition of LSD1 led to a weak hTERT expression, and a robust induction of hTERT mRNA was observed when LSD1 and histone deacetylases (HDACs) were both inhibited. Small interference RNA-mediated depletion of both LSD1 and CoREST, a co-repressor in HDAC-containing complexes, synergistically activated hTERT transcription. In cancer cells, inhibition of LSD1 activity or knocking-down of its expression led to significant increases in levels of hTERT mRNA and telomerase activity. Chromatin immunoprecipitation assay showed that LSD1 occupied the hTERT proximal promoter, and its depletion resulted in elevated di-methylation of histone H3-K4 accompanied by increased H3 acetylation locally in cancer cells. Moreover, during the differentiation of leukemic HL60 cells, the decreased hTERT expression was accompanied by the LSD1 recruitment to the hTERT promoter.Conclusions/SignificanceLSD1 represses hTERT transcription via demethylating H3-K4 in normal and cancerous cells, and together with HDACs, participates in the establishment of a stable repression state of the hTERT gene in normal or differentiated malignant cells. The findings contribute to better understandings of hTERT/telomerase regulation, which may be implicated in the development of therapeutic strategies for telomerase dysregulation-associated human diseases including cancers.