Differentiation Of HL-60 Myeloid Cells Research Articles

WITHDRAWN: MiR-3913-5p is a novel key regulator of differentiation arrest in acute myeloid leukemia

BackgroundMicroRNAs (miRNAs) are a class of small non-coding RNAs that have been shown to play a crucial role in normal hematopoiesis and leukemogenesis. Differentiation arrest is the hallmark of acute myeloid leukemia (AML). However, key miRNAs regulating differentiation arrest in AML remain unclear. MethodsSmall RNA-seq was performed in paired bone marrow samples at initial diagnosis vs. complete remission (CR) and differentially expressed miRNAs (DEMs) were analyzed for each patient. The mRNA levels of significantly upregulated miRNAs in all the patients were assayed during myeloid differentiation induced by all-trans retinoic acid (ATRA) in HL-60 and NB4 cells by Q-PCR. HL-60 and NB4 cells were infected with miR-3913-5p lentivirus (Lenti-miR-3913-5p), and the myeloid differentiation rate was assessed by flow cytometry as well as May–Grünwald Giemsa staining. CCK-8 assay was performed to detect the proliferation of cells. ResultsmiR-3913-5p and other 17 miRNAs were significantly upregulated at initial diagnosis vs. complete remission, and decreased in ATRA-induced myeloid differentiation of HL-60 cells and NB4 cells. Overexpression of miR-3913-5p inhibited myeloid differentiation, while had little effect on proliferation, in HL-60 and NB4 cells. ConclusionsmiR-3913-5p is a novel key regulator of cell differentiation arrest in AML.

Spliceosomal Dysfunction Is a Critical Mediator of IDH2 Mutant Leukemogenesis

Mutations affecting RNA splicing factors are commonly found in leukemias and mutations in SF3B1, SRSF2, and U2AF1 impart specific changes to global splicing. We analyzed RNA-seq data from acute myeloid leukemia (AML) patients from The Cancer Genome Atlas (TCGA) to evaluate for spliceosomal alterations characteristic of mutant SRSF2, SF3B1, and U2AF1 . Although only 1 patient with an SRSF2 mutation was reported in the TCGA AML publication, we identified 19 additional patients to have the characteristic splicing changes of mutant SRSF2 . Mutational analysis of the RNA-seq data identified the SRSF2 proline 95 hotspot mutation in each of these 19 patients (19/178, 11%), making SRSF2 amongst the most commonly mutated genes in the AML TCGA dataset. Interestingly, 47% of SRSF2 mutant patients also had a co-existing IDH2 mutation (and conversely, 50% of IDH2 mutant patients had a co-existing SRSF2 mutation). These data are similar to recent observation that SRSF2 mutations were the most common accompanying mutation in IDH2 mutant myeloid leukemia patients treated on the phase I clinical trial of AG-221.We next sought to understand the functional contribution of SRSF2 mutations to co-existing IDH2 mutations. Overexpression of IDH2 R140Q and IDH2 R172K in Vav -cre Srsf2 P95H or Vav- cre Srsf2 wildtype(WT) backgrounds revealed a clear collaborative effect of mutant IDH2 and mutant Srsf2 (Figure A-B). As early as 4 weeks post-transplant, the peripheral blood of Srsf2 mutant mice transplanted with IDH2 R140Q or IDH2 R172K forms had a substantially greater percentage of GFP+ cells than seen in an Srsf2 WT background. Moreover, these mice exhibited significant myeloid skewing, macrocytic anemia, and thrombocytopenia of greater magnitude than seen with mutant IDH2 expressed in the SRSF2 WT background. IDH2 / Srsf2 double-mutant mice developed a lethal, mixed myelodysplastic/myeloproliferative neoplasm that was serially transplantable (Figure B). Effects from overexpression of mutant IDH2 were recapitulated in mice expressing Idh2 R140Q and Srsf2 P95H from endogenous loci(Mx1 -cre Idh2R140Q/+Srsf2P95H/+ double knock-in mice) (Figure C), providing the first model where spliceosomal gene mutations promote leukemogenesis in vivo .Given prior data identifying inhibition of TET2 as a mediator of IDH2 -mutant leukemogenesis, we evaluated if loss of TET2 might promote transformation of SRSF2 mutant cells. However, deletion of TET2 in an SRSF2 mutant background (Mx1 -cre Tet2fl/flSrsf2P95H/+) was insufficient to promote transformation (Figure D). We next analyzed RNA-seq data from the TCGA, Leucegene, and our own unpublished AML cohort using a novel analysis method to detect unannotated as well as complex splicing events that might explain the basis for the collaboration of mutant IDH2 and SRSF2 . This revealed that IDH2/SRSF2 double-mutant AML had increased aberrant splicing events compared to SRSF2 single-mutant AML and also that IDH2 -mutations are associated with a small but reproducible change in RNA splicing (Figure E). The vast majority of splicing changes affected by SRSF2 and/or IDH2 mutations were a change in cassette exon splicing with SRSF2 mutations promoting inclusion of C-rich containing mRNA sequences, regardless of the presence of concomitant IDH2 mutations (Figure F). One of the most robust mis-splicing events in IDH2/SRSF2 mutant AML was an intron retention event in INTS3 resulting in its reduced mRNA and protein expression (Figure G-H). INTS3 encodes a component of the integrator complex that participates in small nuclear RNA (snRNA) processing. Consistent with this, IDH2 / SRSF2 double-mutant cells exhibited alterations in processing of U2 and U4 snRNAs similar to that seen with direct INTS3 downregulation. Biologically, attenuation of INTS3 expression caused a blockade of myeloid differentiation in HL-60 cells upon ATRA treatment, an effect which was further enhanced in an IDH2 mutant background.These data uncover an important role for spliceosomal gene mutations in the pathogenesis of IDH2 mutant leukemogenesis and identify perturbations in the integrator complex as a novel mediator driving the transformation of IDH2 and SRSF2 mutant cells. The frequent coexistence of IDH2 and SRSF2 mutations underscores the enormous therapeutic potential for modulation of splicing in the ~50% of IDH2 -mutant leukemia patients who also harbor a spliceosomal gene mutation. [Display omitted] DisclosuresDe Botton:Servier: Honoraria; Agios: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Stein:Pfizer: Consultancy, Other: Travel expenses; Novartis: Consultancy, Research Funding; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Celgene Corporation: Consultancy, Other: Travel expenses, Research Funding; Seattle Genetics: Research Funding; GSK: Other: Advisory Board, Research Funding; Constellation Pharma: Research Funding. Peng:H3 Biomedicine Inc.: Employment. Buonamici:H3 Biomedicine Inc.: Employment. Palacino:H3 Biomedicine Inc.: Employment. Smith:H3 Biomedicine Inc.: Employment. Thompson:Merck: Membership on an entity's Board of Directors or advisory committees; Charles River Laboratories: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Founder. Levine:Roche: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding; Celgene: Research Funding.

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.

Styryl quinazolinones and its ethynyl derivatives induce myeloid differentiation.

The tumor suppressor transcription factor CCAAT enhancer-binding protein α (C/EBPα) expression is downregulated in myeloid leukemias and enhancement of C/EBPα expression induces granulocytic differentiation in leukemic cells. Previously we reported that Styryl quinazolinones induce myeloid differentiation in HL-60 cells by upregulating C/EBPα expression. To identify more potent molecule that can induce leukemic cell differentiation we synthesized and evaluated new series of styryl quinazolinones, ethynyl styryl quinazolinones, styryl quinolinones and thienopyrimidinones. Thienopyrimidinones were found toxic and styryl quinolinones were found inactive. Ethynyl styryl quinazolinone 39 and styryl quinazolinone 5 were found active on par with the earlier reported analogues 1 and 2 suggesting that the 5-nitro furan-2-yl styryl quinazolinones find a real promise in leukemic cell differentiation. The improved potency of 5 suggested that further modifications in the 5-nitro furan-2-yl styryl quinazolinones can be at the phenyl substitution at the 3-position of the quinazolinone ring apart from the 5-position of the heteroaryl ring.

PS978 DISCOVERY OF NOVEL C/D BOX SNORNAS THAT ARE REQUIRED FOR LEUKEMIA CELL PROLIFERATION

Background:The vast majority of the human genome is transcribed, but the function of many transcripts remain obscure. It is likely, that cancer specific transcripts exist that are overlooked by conventional pipeline analysis.Aims:We tried to identify novel non coding RNA transcripts with C/D box snoRNA properties based on sequence homology. We specifically analysed AML patient samplesMethods:To understand the role of snoRNAs in leukemogenesis, we analyzed the snoRNA expression pattern in an initial cohort of 63 AML patients (Zhou, 2017) and an additional cohort of 90 AML patients. Abundant regions with coverage were annotated using snoRNABase (LBME), the recently published updated snoRNAome (Jorjani, 2016) and tracks from ENSEMBL, UCSC, RNAcentral and DASHR databases. Regions without annotation were analyzed for C/D box motifs to discover putative C/D box snoRNAs. For further analysis only regions with at least one reads per million (RPM) in at least one patient sample were included.Results:Next generation sequencing data revealed that 248 of the 269 known C/D box snoRNAs (92%) were expressed in one or more patient samples. For the H/ACA box snoRNAs we could identify 103 of 112 (92%). In addition to this established functionally well annotated core‐set of C/D box snoRNA we confirmed another set of 76 C/D box snoRNA to be expressed in AML patient samples. These 76 snoRNA recently have been described as novel C/D‐box snoRNA based on the analysis of ENCODE small RNA‐seq data. Importantly, our in silico analysis identified 45 novel putative C/D box snoRNAs which had not been described so far. All of these putative C/D box snoRNAs contain the box C/D motifs and were mostly located in introns of known genes. In further analyses, we observed increased expression of putative snoRNAs was found in patients with intermediate molecular risk (7 snoRNAs, p≤0.05) and in patients with a poor response to chemotherapy (9 snoRNAs, p≤0.05). The novel snoRNAs were more prominently expressed in in NPM1 wildtype AML compared to NPM1 mutant AML. Overall, 13 putative C/D box snoRNA were downregulated during myeloid differentiation of HL60 cells. To study the function of the snoRNA we performed a CRISPR/Cas9 knockout of five regions in Kasumi1 and U937 cell lines. The knockout of four out of five novel snoRNAs impaired proliferation and colony growth.Summary/Conclusion:Multiple novel C/D box snoRNAs were identified from primary AML specimens. These show a specific expression pattern in myeloid differentiation in distinct AML subtypes. The functional relevance of these snoRNAs suggests that so far overlooked non coding RNA sequences are of high importance in AML.

Elevated EGR2 Expression Provides a Potential Link Between Cell Cycle Arrest and Induced Differentiation in Myeloid Progenitor Cells

Similar to many immature cell types, myeloid progenitor cells need to exit cell cycle to undergo terminal differentiation, but the mechanism linking the two is still unclear. Elucidating this mechanism could lead to the development of new differentiation therapies to treat myeloid leukemia. Recent studies have suggested that the processes regulating myeloid differentiation and cell cycle progression together constitute a positive feedback loop where each process reciprocally affects the other. To study the relationship between these processes, we examined early cellular and molecular events associated with induced differentiation of the HL60 human promyelocytic leukemia cells. We treated HL60 cells with 3 classical inducers of differentiation (vitamin D3 analog EB1089 (EB), all-trans retinoic acid (ATRA), and dimethyl sulfoxide (DMSO)), along with PD0332991 (PD), a selective cyclin D-dependent kinase 4/6 inhibitor that caused G1-phase-specific cell-cycle arrest. We evaluated differentiation of the treated cells by flow cytometric analysis of CD11b (integrin αM) and CD71 (transferrin receptor) expression. In untreated HL60 cells, a baseline subset of 3-5% of cells exhibits a differentiated, CD11b+CD71- phenotype. Exposure to the various inducers revealed a progressive increase in the percentage of CD11b+CD71- cells with time, such that by day 4 of treatment, it has increased to 50-90% in the treated samples, indicating that all 4 agents tested were effective in inducing myeloid differentiation. To understand how differentiation induced by each agent affects cell cycle progression, the cell cycle status of the induced cells were evaluated by a BrdU-incorporation assay after a 30-minute pulse of BrdU labeling. Uninduced cells exhibited a baseline cell cycle phase distribution of 64%-28%-8% (G1-S-G2/M phases). After 1 day of induction, EB-treated sample showed no changes in the distribution (58%-33%-9%), but ATRA, DMSO and PD-treated samples showed significant changes, with an increase of cell numbers in G1 phase and decrease in S phase (74%-18%-8%, 79%-13%-8%, and 93%-4%-3%, respectively). These results reveal that an early induction of G1 arrest was caused by treatment with ATRA, DMSO and PD, but not EB, and that the cell cycle arrest occurred before major changes in the myeloid phenotype were observed. To determine how the cell cycle perturbation relates to changes in the underlying genetic regulatory network, we examined by quantitative RT-PCR analysis the expression of several transcription factors associated with myeloid differentiation. PU.1 and CEBPA were found to be expressed at high levels but these levels did not change upon treatment with the inducing agents. Similarly, the expression levels of GFI1 and EGR1 did not change significantly with induction. In contrast, the expression level of EGR2 (Early Growth Response 2) was found to be low initially but became elevated upon treatment with 3 of the 4 inducers. EGR2 is a zinc finger transcription factor implicated in the control of a switch between pro- and anti-proliferation pathways. EGR2 has been shown to regulate the transition between differentiation states of Schwann cells, induction of anergic and regulatory T cells, growth and survival of osteoclasts, and proliferation and apoptosis of acute myeloid leukemia blasts. We found that EGR2 expression, after 1 day of treatment with ATRA, DMSO or PD, was increased by 5.2 ± 0.9, 7.6 ± 1.9, 5.8 ± 0.9 folds, respectively, whereas treatment with EB led to no significant change (1.5 ± 0.2 fold). We evaluated whether simultaneous treatment of the cells with 2 inducers would result in an additive effect. Treatment of HL60 cells with a combination of ATRA/DMSO, ATRA/PD, or DMSO/PD increased the percentage of CD11b+CD71- cells to 55%, 70% and 25% after just 1 day of treatment. In line with the enhanced phenotypic effect, the expression level of EGR2 was further elevated to 7.7 ± 1.4, 15.4 ± 3.5, and 11.3 ± 3.4 folds, respectively, when the cells were treated with the above inducer combinations, indicating a tight association between EGR2 expression and the phenotypic effect. In summary, our data suggest that elevated expression of EGR2 is an early event in the induction of myeloid differentiation in HL60 cells. Because of its known role in cell cycle regulation, EGR2 could function as a mechanistic link between cell cycle arrest and induced differentiation in myeloid progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Free LIF receptor α-chain distal cytoplasmic motifs enhance Jak2-independent STAT3 phosphorylation and induce differentiation in HL-60 cells

The leukemia inhibitory factor (LIF) affects multiple types of leukemia cells invitro through the functional LIF receptor (LIFR), which comprises a complex of the LIFRα-chain (LIFR α) and gp130. As Jak2/STAT3 has been proven to be a significant mediator in the LIF-induced differentiation of promyeloid leukemia cells, we constructed a recombinant vector, pcDNA3.0-CT3 (containing the structurally conserved triple YXXQ motifs of LIFR α, termed LIFR α-CT3), and its specific tyrosine-mutated counterpart, pcDNA3.0-MUT, to determine the sites and examine the corresponding mechanisms involved in STAT3 phosphorylation. We found that the triple YXXQ motifs of LIFR α-CT3 are capable of up-regulating phosphorylated levels of STAT3 in a Jak2-independent manner prior to the induction of myeloid differentiation by LIFR α-CT3 in the human promyeloid cell line HL-60. By specifically blocking Jak2 using the AG-490 inhibitor, we observed that the LIFR α-CT3 group of HL-60 cells still demonstrated up-regulation of phosphorylated STAT3 and this up-regulation could result in the myeloid differentiation of HL-60 cells. These results may shed light on acute promyeloid leukemia therapy in future clinical practice.

Ellagic acid, a natural polyphenolic compound, induces apoptosis and potentiates retinoic acid-induced differentiation of human leukemia HL-60 cells

All-trans retinoic acid (ATRA) is a standard drug used for differentiation therapy in acute promyelocytic leukemia. To potentiate this therapy, we examined the effect of ellagic acid (EA), a natural polyphenolic compound with antiproliferative and antioxidant properties, on the growth and differentiation of HL-60 acute myeloid leukemia cells. EA was found to induce apoptosis, which was blocked by pan-caspase inhibitor, Z-VAD-FMK. EA activated the caspase-3 pathway and enhanced the expressions of myeloid differentiation markers (CD11b, MRP-14 protein, granulocytic morphology) induced by ATRA treatment. These results indicate that EA is a potent apoptosis inducer and also effectively potentiates ATRA-induced myeloid differentiation of HL-60 cells.

Deamino-hydroxy-phoslactomycin B, a biosynthetic precursor of phoslactomycin, induces myeloid differentiation in HL-60 cells

During the screening for novel differentiation inducers, we found that a culture broth of Streptomyces sp. HK-803 induced myeloid differentiation of HL-60 cells. The active substance was identified as deamino-hydroxy-phoslactomycin B (HPLM) by mass spectrometry, and synthesized HPLM also induced the differentiation of HL-60 cells. HPLM showed greater inhibition of protein phosphatase 2A (PP2A) activity than phoslactomycin B (PLMB); however, PLMB and okadaic acid did not induce differentiation. Moreover, treatment with ATRA and 1α, 25(OH) 2D 3 induced retinoic acid receptor-β and 1α, 25(OH) 2 D 3 24-hydroxylase, respectively, whereas HPLM did not, suggesting that HPLM is a novel differentiation inducer.

Inverse Correlation of NM23 Expression with Lysophosphatidic Acid Receptor EDG2/lpa1 Expression of Human Leukemia Cells during Myeloid Differentiation Induced by All-Trans Retinoic Acid

Overexpression of the nm23 gene is found in many hematological malignancies, and predicts poor treatment outcome. Because the amount of intracellular NM23-H1 protein is inversely correlated with differentiation and exogenous over expression of the nm23-H1 in myeloid leukemia cells reduced the sensitivity to induction of myeloid differentiation, nm23-overexpression is considered to function as a differentiation-suppressor. However, the molecular mechanism of this phenomenon is unknown. Recently, lysophosphatidic acid (LPA) receptor EDG2/lpa1 was identified as a gene down-regulated by nm23-H1 and associated with cell motility/metastasis suppressor functions in breast cancer cells. Although the clinical significance of nm23-H1 overexpression in leukemia is completely opposite to that in breast cancer, there might be a common molecular mechanism between the metastasis-suppressing activity and differentiation-inhibiting activity of nm23-H1 overexpression, because many functional characteristics of macrophages/neutrophils are similar to those of motile and metastatic tumor cells. Here, we examined the EDG2/lpa1 expression level and its association with NM23 expression level and myeloid differentiation of leukemia cells. EDG2 and NM23 proteins were expressed at different levels in all leukemia cells tested (HL-60, NB4, THP-1, U937, K562, HEL, BALM-1, BALM-3, MOLT4, Jurkat). During all-trans retinoic acid (ATRA)-induced myeloid differentiation of HL-60 cells, the NM23 expression decreased and EDG2 expression inversely increased. Similar effects of ATRA were observed in myeloid differentiation of NB4 and THP-1 cells. NM23 expression levels and EDG2 expression levels modulated by ATRA treatment were inversely correlated (Spearman's correlation coefficient, rs = −0.7538, p = 0.0237). However, these correlations were not found in the erythroid differentiation of ATRA-treated HEL and K562 cells. Cell motility is required for myeloid differentiation; therefore, there might be a common mechanism, the inhibition of cell motility thorough EDG2 down-regulation by NM23-H1 overexpression, between differentiation-inhibiting activity in leukemia cells and motility-inhibiting activity in breast cancer cells. The discrepancy in the clinical significance of NM23-H1 overexpression between leukemia and breast cancer might be resolved.

Inhibiting the platelet derived growth factor receptor increases signs of retinoic acid syndrome in myeloid differentiated HL-60 cells

PDGFR inhibitors are successfully used in a number of cancer treatments. The standard treatment for acute promyelocytic leukemia (APL) involves differentiation therapy with retinoic acid (RA). However, the relapse rates are significant. In the present work we evaluated the effects of RA therapy in the presence of PDGFR inhibitor, AG1296. Adding AG1296 with RA increased secretion of TNF-α, IL-8, and MMP-9 expression. This treatment induced higher levels of ICAM-1 endothelial cell expression, and increased cellular mobility. Inhibiting PDGFR enhanced RA-induced expression of integrin. Integrin ligand increased differentiation markers CD11b, inducible oxidative metabolism and PDGFR-β phosphorylation. While the neutrophil–endothelial cell interactions are strengthened by the combined treatment, the endothelium-substratum interactions are weakened, a situation common in RAS.

Src Kinase Inhibitors Enhance ATRA-Mediated Differentiation of Myeloid Cells.

Myeloid leukemias are characterized by a blockade in differentiation resulting in accumulation of proliferating progenitor cells and a lack of mature granulocytes and monocytes. In acute promyelocytic leukemia (APL), a subtype of AML, treatment with all-trans retinoic acid (ATRA) has been shown to overcome this blockade in differentiation and is the current standard of care for APL patients. However, other forms of myeloid leukemia show no response to ATRA. Src family kinases (SFK) have been shown to be activated during normal myeloid development by both proliferation-inducing cytokines, such as IL-3, and differentiation-promoting factors, such as G-CSF. To elucidate the role of src family kinases during myeloid differentiation, we examined the impact of SFK inhibitors, PP1 and PP2 on ATRA-mediated differentiation of myeloid cell lines. Interestingly, PP1 potentiated ATRA-mediated myeloid differentiation of HL-60 cells, inducing a two-fold increase in differentiated cells at 72 hours as determined by CD11b staining, NBT reduction and morphological analysis. To determine whether enhancement of ATRA-mediated differentiation was specific for PP1, or could be promoted by a different src family kinase inhibitor, we evaluated the impact of PP2. PP2 was found to exhibit similar effects on ATRA-induced myeloid differentiation of HL-60 cells. Additionally, both PP1 and PP2 enhanced ATRA-induced monocytic differentiation of U937 cells and granulocytic differentiation of NB-4 cells. Interestingly, PP1 was found to enhance ATRA-induced differentiation even when added 24 hours after addition of ATRA. The impact of PP1 on ATRA-mediated myeloid differentiation was dependent on signaling via the MEK/ERK pathway since pre-treatment with U0126, a MEK-1/-2 inhibitor, attenuated myeloid differentiation induced by the combination of RA and PP1. Reporter assays utilizing an RARE-luciferase construct indicate that PP1 and PP2 do not enhance ATRA-mediated differentiation by promoting the transcriptional activity of the retinoic acid receptor (RAR). Together, our results demonstrate that PP1 and PP2 potently enhance ATRA-induced myeloid differentiation of AML cell lines and suggest that src inhibition in combination with ATRA may be a useful treatment for AML. Studies evaluating the impact of src inhibition on ATRA-mediated differentiation of primary AML cells are currently underway.

MicroRNA hsa-mir-16 Contributes to Regulation of Myeloid Differentiation of Human CD34+ Cells.

In a large microRNA-array and bioinformatics study, we determined all of the microRNAs (miRs) expressed by human CD34+ hematopoietic stem-progenitor cells (HSPCs) from bone marrow and G-CSF mobilized blood. When we combined miR expression data, mRNA expression data fro a previous study (Georgantas et al, Cancer Research 64:4434), and data from various published mir-target prediction algorithms, we were able to bioinformaticly predict the actions of miRs within the hematopoietic system. MicroRNA hsa-mir-16 was highly expressed in CD34+ HSPCs, and was predicted to target several HSPC-expressed mRNAs (CXCR4, HoxB7, Runx-1, ETS-1, and Myb) that encode proteins known to be critically involved specifically in myelopoiesis within the hematopoietic system. We first confirmed that protein expression from each of these putative target mRNAs was in fact regulated by mir-16. The 3′UTR sequence from each of these mRNAs was cloned behind a luciferase reporter. Each reporter construct was transfected into K562 cells, which strongly express mir-16. In all cases, protein expression from the predicted target mRNA was greatly reduced in K562 cells, as compared to controls. As a first determination of mir-16's function in hematopoietic cells, HL60 and K562 cells were transduced with hsa-mir-16 lentivirus, then treated with various chemical differentiation inducers. As was predicted by bioinformatics, hsa-mir-16 halted myeloid differentiation of HL60 cells, but did not affect megakaryocytic differentiation or erythroid differentiation of K562 cells. These initial findings suggest that mir-16 is a specific negative regulator of myelopoiesis. We are currently evaluating the effects of mir-16 on normal human CD34+ cells by in vitro CFC and suspension culture assays, as well as in vivo by transplantation of hsa-mir-16 lentivirus transduced cells in immunodeficient mice.

Induction of Lactoferrin Gene Expression in Myeloid or Mammary Gland Cells by Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax: Implications for Milk-Borne Transmission of HTLV-1

Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia, is transmitted vertically via breastfeeding. We have previously demonstrated that lactoferrin, a major milk protein, enhances HTLV-1 replication, at least in part by upregulating the HTLV-1 long terminal repeat promoter. We now report that HTLV-1 infection can induce lactoferrin gene expression. Coculture with HTLV-1-infected MT-2 cells increased the levels of lactoferrin mRNA in myeloid-differentiated HL-60 cells, as well as MCF-7 cells, models of two probable sources (neutrophils and mammary epithelium) of lactoferrin in breast milk. MT-2 cell coculture could be replaced with cell-free culture supernatants of MT-2 cells to exert the same effect. Furthermore, extracellularly administered Tax protein also induced lactoferrin gene expression at physiologically relevant concentrations. In transient-expression assays, Tax transactivated the lactoferrin gene promoter in HL-60 or MCF-7 cells. Experiments with Tax mutants, as well as site-directed mutants of the lactoferrin promoter reporters, indicated that the NF-kappaB transactivation pathway is critical for Tax induction of the lactoferrin gene promoter activity in myeloid-differentiated HL-60 cells, but not in MCF-7 cells. These results suggest that HTLV-1 infection may be able to induce expression of lactoferrin in a paracrine manner in the lactic compartment. Our findings, in conjunction with our previous study, implicate that mutual interaction between HTLV-1 and lactoferrin would benefit milk-borne transmission of this virus.

The synthetic furanonaphthoquinone induces growth arrest, apoptosis and differentiation in a variety of leukaemias and multiple myeloma cells

2-methyl-naphtho[2,3-b]furan-4,9-dione (FNQ3), a synthetic analogue of the quinone kigelinone, has demonstrated a real potential for use in the treatment of a variety of solid tumours. Unlike other quinones, such as mitomycin-C and adriamycin, the cytotoxicity of FNQ3 is often 10- to 14-fold more potent towards the tumour cells than their normal counterparts. We report, for the first time, that the drug had activity against a broad spectrum of leukaemias and multiple myeloma cells. It decreased the growth of acute myeloid leukaemia (AML) and multiple myeloma cell lines in a dose-dependent fashion (50% inhibitory concentration approximately 1.25 microg/ml against most of the leukaemia cell lines). This dose apparently initiated mitochondrial collapse as measured by depolarisation of the mitochondrial membrane. FNQ3 potentiated the differentiation of HL-60 myeloid cells in the presence of either 1alpha, 25(OH)(2) dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or all-trans-retinoic acid (ATRA). FNQ3 inhibited the proliferation of primary AML cells while inducing apoptosis. Eleven of 14 (79%) AML marrow samples had a prominent decrease in their clonogenic growth when cultured in the presence of the drug. In summary, this drug has growth inhibitory, apoptotic and differentiative effects against myeloid leukaemias and multiple myeloma cells. FNQ3 may represent a new therapeutic approach to these malignancies.

MicroRNA-196 inhibits HOXB8 expression in myeloid differentiation of HL60 cells

MicroRNAs (miRNAs) are endogenous small RNAs (approximately 21-25 nts) that can regulate gene expression at a post-transcriptional level. Recently a large number of miRNAs have been identified from plants and animals. In addition, it has been reported that these small RNAs can participate in cell differentiation of developmental timing. In this study, we show that microRNA-196 (miR-196) inhibits HOXB8 expression in myeloid differentiation of HL 60 cells. MiR-196 has perfect complementarity with target sequence of 3'-UTR in HOXB8 mRNA. Exogenous expressed miR-196 repressed expression of HOXB8 via cleaving mRNAs. Moreover miR-196 enhanced myeloid differentiation of HL60 cells. These results suggest that miR-196 participates in myeloid differentiation of HL60 cells via regulation of HOXB8 expression.

Human EVI9, a Homologue of the Mouse Myeloid Leukemia Gene, Is Expressed in the Hematopoietic Progenitors and Down-Regulated during Myeloid Differentiation of HL60 Cells

Evi9, a common site of retroviral integration in BXH2 murine myeloid leukemias, encodes a C2H2 zinc finger protein and is overexpressed in these leukemic cells. To investigate a possible role of EVI9 in the human hematopoietic system, we isolated the cDNA clone of the human homologue. Human EVI9, located on the chromosome 2p13 region, contains an open reading frame of 797 amino acids that is 98.7% identical to the mouse protein. RT-PCR analysis of purified human hematopoietic cells showed that EVI9 is expressed in CD34-positive myeloid precursors, B cells, monocytes, and megakaryocytes, but only weakly in T lymphocytes, suggesting that EVI9 may play an important role in hematopoiesis. Furthermore, EVI9 was down-regulated during myeloid differentiation of HL60 cells induced by all-trans-retinoic acid, whereas the expression remained during monocytic differentiation induced by phorbol 12-myristate 13-acetate. These results indicate a distinct role for EVI9 in human hematopoietic cells and suggest that EVI9 may cause leukemia through inhibition of myeloid differentiation.

Expression of purinergic receptors (ionotropic P2X1–7 and metabotropic P2Y1–11) during myeloid differentiation of HL60 cells

The expression of human purinergic P2 receptors (P2X1–7 and P2Y1–11) as well as the ecto-enzymes apyrase (CD39) and 5′-nucleotidase (CD73) was investigated on the nucleic acid level during granulocytic and monocytic differentiation of HL60 cells and on peripheral human blood leukocytes. RT-PCR and dot-blot hybridization assays indicated that mRNA transcripts of all analyzed P2 receptors apart from the P2X3 receptor were expressed during myeloid development of HL60 cells, showing a distinct regulation during the course of differentiation. In blood leukocytes, transcripts of P2X5, P2X7 and all P2Y receptors, except for P2Y6, receptor were found. CD39 and CD73 showed a marked upregulation during myeloid maturation. Functional analysis of P2 receptor-mediated intracellular Ca 2+-increase after stimulation with ATP revealed no change during granulocytic differentiation, but showed a strong attenuation in both potency and efficacy during monocytic development of HL60 cells.

Stimulation of phospholipase C-β2 by Rho GTPases

This chapter describes the stimulation of phospholipase C-β 2 by Rho guanosine 5'-triphosphates (GTPases). It has been shown that the cytosolic preparations of myeloid differentiated HL-60 cells and of bovine neutrophils contain a soluble phospholipid-specific phospholipase C (PLC) that is activated by the poorly hydrolyzable GTP analog GTPγS. The chapter discusses the purification of recombinant PLC-β 2 Δ, the purification of native and recombinant heterodimers of LyGD1 and Rho GTPases, and the in vitro assays used to measure GTPγS binding to and stimulation of PLC- β 2 by Rho GTPases. The purification method described in this chapter not only proves to be satisfactory in studying PLC β 2 stimulation by recombinant LyGDI-Rho heterodimers but also enables the crystallization of LyGDI-Rac2 dimer to analyze its structure by X-ray crystallograph. The chapter concludes with a discussion of the expression of recombinant PLC-β 2 Δ in insect cells.

Formyl Peptide Receptor Signaling in HL-60 Cells through Sphingosine Kinase

Sphingosine-1-phosphate (SPP) produced from sphingosine by sphingosine kinase has recently been reported to act as intracellular second messenger for a number of plasma membrane receptors. In the present study, we investigated whether the sphingosine kinase/SPP pathway is involved in cellular signaling of the Gi protein-coupled formyl peptide receptor in myeloid differentiated human leukemia (HL-60) cells. Receptor activation resulted in rapid and transient production of SPP by sphingosine kinase, which was abolished after pertussis toxin treatment. Direct activation of heterotrimeric G proteins by AlF4- also rapidly increased SPP formation in intact HL-60 cells. In cytosolic preparations of HL-60 cells, sphingosine kinase activity was stimulated by the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate). Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine did not affect phospholipase C stimulation and superoxide production but markedly inhibited receptor-stimulated Ca2+ mobilization and enzyme release. We conclude that the formyl peptide receptor stimulates through Gi-type G proteins SPP production by sphingosine kinase, that the enzyme is also stimulated by direct G protein activation, and that the sphingosine kinase/SPP pathway apparently plays an important role in chemoattractant signaling in myeloid differentiated HL-60 cells.