Human Myelomonocytic Leukemia Cells Research Articles

Design, optimization, production and activity testing of recombinant immunotoxins expressed in plants and plant cells for the treatment of monocytic leukemia

ABSTRACTAntibody–drug conjugates (ADCs) can improve therapeutic indices compared to plain monoclonal antibodies (mAbs). However, ADC synthesis is complex because the components are produced separately in CHO cells (mAb) and often by chemical synthesis (drug). They are individually purified, coupled, and then the ADC is purified, increasing production costs compared to regular mAbs. In contrast, it is easier to produce recombinant fusion proteins consisting of an antibody derivative, linker and proteinaceous toxin, i.e. a recombinant immunotoxin (RIT). Plants are capable of the post-translational modifications needed for functional antibodies and can also express active protein toxins such as the recombinant mistletoe lectin viscumin, which is not possible in prokaryotes and mammalian cells respectively. Here, we used Nicotiana benthamiana and N. tabacum plants as well as tobacco BY-2 cell-based plant cell packs (PCPs) to produce effective RITs targeting CD64 as required for the treatment of myelomonocytic leukemia. We compared RITs with different subcellular targeting signals, linkers, and proteinaceous toxins. The accumulation of selected candidates was improved to ~ 40 mg kg−1 wet biomass using a design of experiments approach, and corresponding proteins were isolated with a purity of ~ 80% using an optimized affinity chromatography method with an overall yield of ~ 84%. One anti-CD64 targeted viscumin-based drug candidate was characterized in terms of storage stability and cytotoxicity test in vitro using human myelomonocytic leukemia cell lines. We identified bottlenecks in the plant-based expression platform that require further improvement and assessed critical process parameters that should be considered during process development for plant-made RITs.

CK2 Inhibitor CX4945 Shows Strong In Vivo Anti Leukemia Effect in AML Via Augmented Ikaros-Mediated Regulation of Global Epigenetic Landscape

Over-expression of Casein Kinase II (CK2) pro-oncogenic kinase in Acute Myelogenous Leukemia (AML) is associated with poor prognosis. Inhibition of CK2 by siRNAs or with the specific inhibitor, CX-4945, shows strong cytotoxic effects. CK2 is a ubiquitous, constitutively active serine/ threonine kinase which has been implicated in leukemia progression via multiple mechanisms. One of the well-established mechanism is CK2 mediated phosphorylation and impairment of tumor suppressor function of Ikaros transcription factor. Impaired function of Ikaros has been associated with the development of leukemia including AML. In B-cell lymphoblastic leukemia, CK2 inhibition has been shown to exert a therapeutic effect via restoration and/or enhancement of the tumor suppressor activity of the Ikaros protein. The mechanism of therapeutic action of CK2 inhibition in AML is largely unknown and in vivo efficacy CK2 inhibitors in AML has never been established. Here we report anti-leukemia effect of CX-4945 in preclinical models of AML and demonstrate mechanism of action of CK2 inhibitor via restoration of Ikaros driven global regulation of epigenetic landscape. In order to study the effect of CK2 inhibition on Ikaros function in AML, we used U937 - a human myelomonocytic leukemia cell line with high baseline CK2 expression. U937 cells were treated with specific CK2 inhibitor, CX4945 for 72 hours. CK2 activity and Ikaros phosphorylation was measured using CK2 kinase assay and in vivo labeling - radio immunoblot assay. Results showed significant decrease in CK2 activity and Ikaros phosphorylation with no change in overall expression of the Ikaros protein. Expression analysis using RNA sequencing showed that treatment with CX-4945 caused significant upregulation of genes controlling immunity, and inflammation; and downregulation of genes involved in nucleic acid metabolism, RNA processing, and translation. Analysis of global genome-wide Ikaros occupancy using Chromatin Immunoprecipitation followed by next generation sequencing (ChIP-seq) of CX4945 treated U937 cells demonstrated that CX-4945 treatment significantly increased the number of Ikaros binding sites as well as increased peak strength while minimally re-distributing Ikaros' global genomic occupancy. While increased binding to the Promoter, Gene Body, and Gene Desert elements in CX-4945-treated cells was similar, the increase in Ikaros' DNA binding to enhancers was particularly pronounced. Further analysis showed that enhanced Ikaros DNA binding following treatment with CX-4945, directly induces formation of de novo enhancers. In order to test whether enhanced DNA binding of Ikaros is accompanied by augmented Ikaros function in the global epigenetic regulation of gene expression. We determined and compared chromatin accessibility of U937 cells before and after treatment with CX-4945, using ATAC-seq. Results indicate that Ikaros-induced de novo open chromatin at distal regulatory regions controls genes involved in the negative regulation of biological processes and cellular metabolism. Overall, these data demonstrate that, augmented Ikaros DNA-binding following CK2 inhibition resulted in 1) Ikaros' pioneering activity, 2) Ikaros' ability to induce the de novo formation of enhancers and super-enhancers, and 3) Ikaros' ability to induce the de novo formation of active enhancers and to activate poised enhancers. Together, these data uncover novel Ikaros functions in regulating the epigenetic landscape and identifies CK2 as a critical regulator of Ikaros activity. Next, we treated AML xenograft model of luciferase labelled U937 cells with CX4945 via oral gavage at dose 200mg/kg/day for 3 weeks and demonstrated significantly lower leukemia burden in treated group as measured by decreased bio-luminescence imaging. In summary, these results demonstrate for the first time that CK2 inhibitor, CX-4945 has strong anti-leukemia effect in AML preclinical models. One of the mechanisms by which CX4945 exert a therapeutic effect in AML involves enhancing Ikaros' function as regulator of global epigenomic landscape. These results provide strong mechanistic basis to develop novel targeted combination therapies using CK2 inhibitors for treatment of AML. Further studies evaluating combination therapies using patient derived xenograft (PDX) models of AML are underway. Disclosures Payne: Elf Zone, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Baicalin Augments Hyperthermia-Induced Apoptosis in U937 Cells and Modulates the MAPK Pathway via ROS Generation

Background/Aims: Hyperthermia is a widely used therapeutic tool for cancer therapy and a well-known inducer of apoptosis. Although the flavonoid compound baicalin (BCN) is a potent anticancer agent for several human carcinomas, it is less potent in the human U937 myelomonocytic leukemia cell line. To explore any enhancing effects of BCN on hyperthermia-induced apoptosis, this study investigated the combined effects and apoptotic mechanisms of hyperthermia and BCN in U937 cells. Methods: U937 cells were heat treated at 44ºC for 12 min with or without pre-treatment with BCN (10-50 µM) and then incubated for 6 h at 37 ºC with 5% CO2 and 95% air. Cell viability was analyzed by Trypan blue exclusion assay. Apoptosis was examined by DNA fragmentation, fluorescence microscopy and flow cytometry. Generation of mitochondrial trans-membrane potential (MMP), mitochondrial calcium, and reactive oxygen species (ROS) was also detected by flow cytometry. The expression of proteins related to apoptosis and signaling pathways was determined by western blotting. Results: Hyperthermia alone did not reduce cell viability or induce notable levels of apoptosis, but combined hyperthermia and BCN treatment markedly augmented apoptosis by upregulating proapoptotic proteins and suppressing antiapoptotic proteins, culminating in caspase-3 activation. Mitochondrial transmembrane potential was significantly decreased, and generation of reactive oxygen species (ROS) and suppression of antioxidant enzymes were marked. Furthermore, with the combined treatment, the phosphorylated forms of JNK and p38 showed increased expression, whereas AKT was dephosphorylated. JNK-IN-8 (a JNK inhibitor) and NAC (a ROS scavenger) abrogated the apoptotic effects of the combined treatment, significantly protecting the cells and indicating the involvement of high ROS generation and the MAPK pathway in the underlying molecular mechanism. Conclusion: This study provides compelling evidence that hyperthermia, in combination with BCN, is a promising therapeutic strategy for enhancement of apoptosis and suggest a promising therapeutic approach for cancer.

ChemInform Abstract: Aplysiasecosterol A: A 9,11‐Secosteroid with an Unprecedented Tricyclic γ‐Diketone Structure from the Sea Hare Aplysia kurodai.

AbstractThe title compound shows moderate cytotoxicity against human myelomonocytic leukemia cell line HL‐60.

Anti-proliferative activity of Monensin and its tertiary amide derivatives.

New tertiary amide derivatives of polyether ionophore Monensin A (MON) were synthesised and their anti-proliferative activity against cancer cell lines was studied. Very high activity (IC50=0.09 μM) and selectivity (SI=232) of MON against human biphenotypic myelomonocytic leukemia cell line (MV4-11) was demonstrated. The MON derivatives obtained exhibit interesting anti-proliferative activity, high selectivity index and also are able to break the drug-resistance of cancer cell line.

System-wide analysis of the transcriptional network of human myelomonocytic leukemia cells predicts attractor structure and phorbol-ester-induced differentiation and dedifferentiation transitions.

We present a system-wide transcriptional network structure that controls cell types in the context of expression pattern transitions that correspond to cell type transitions. Co-expression based analyses uncovered a system-wide, ladder-like transcription factor cluster structure composed of nearly 1,600 transcription factors in a human transcriptional network. Computer simulations based on a transcriptional regulatory model deduced from the system-wide, ladder-like transcription factor cluster structure reproduced expression pattern transitions when human THP-1 myelomonocytic leukaemia cells cease proliferation and differentiate under phorbol myristate acetate stimulation. The behaviour of MYC, a reprogramming Yamanaka factor that was suggested to be essential for induced pluripotent stem cells during dedifferentiation, could be interpreted based on the transcriptional regulation predicted by the system-wide, ladder-like transcription factor cluster structure. This study introduces a novel system-wide structure to transcriptional networks that provides new insights into network topology.

Gene Regulatory Scenarios of Primary 1,25-Dihydroxyvitamin D3 Target Genes in a Human Myeloid Leukemia Cell Line

Genome- and transcriptome-wide data has significantly increased the amount of available information about primary 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) target genes in cancer cell models, such as human THP-1 myelomonocytic leukemia cells. In this study, we investigated the genes G0S2, CDKN1A and MYC as master examples of primary vitamin D receptor (VDR) targets being involved in the control of cellular proliferation. The chromosomal domains of G0S2 and CDKN1A are 140–170 kb in size and contain one and three VDR binding sites, respectively. This is rather compact compared to the MYC locus that is 15 times larger and accommodates four VDR binding sites. All eight VDR binding sites were studied by chromatin immunoprecipitation in THP-1 cells. Interestingly, the site closest to the transcription start site of the down-regulated MYC gene showed 1,25(OH)2D3-dependent reduction of VDR binding and is not associated with open chromatin. Four of the other seven VDR binding regions contain a typical DR3-type VDR binding sequence, three of which are also occupied with VDR in macrophage-like cells. In conclusion, the three examples suggest that each VDR target gene has an individual regulatory scenario. However, some general components of these scenarios may be useful for the development of new therapy regimens.

Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l

AbstractThe t(6;11)(q27;q23) is a recurrent chromosomal rearrangement that encodes the MLLAF6 fusion oncoprotein and is observed in patients with diverse hematologic malignancies. The presence of the t(6;11)(q27;q23) has been linked to poor overall survival in patients with AML. In this study, we demonstrate that MLL-AF6 requires continued activity of the histone-methyltransferase DOT1L to maintain expression of the MLL-AF6-driven oncogenic gene-expression program. Using gene-expression analysis and genome-wide chromatin immunoprecipitation studies followed by next generation sequencing, we found that MLL-fusion target genes display markedly high levels of histone 3 at lysine 79 (H3K79) dimethylation in murine MLL-AF6 leukemias as well as in ML2, a human myelomonocytic leukemia cell line bearing the t(6;11)(q27;q23) translocation. Targeted disruption of Dot1l using a conditional knockout mouse model inhibited leukemogenesis mediated by the MLL-AF6 fusion oncogene. Moreover, both murine MLL-AF6–transformed cells as well as the human MLL-AF6–positive ML2 leukemia cell line displayed specific sensitivity to EPZ0004777, a recently described, selective, small-molecule inhibitor of Dot1l. Dot1l inhibition resulted in significantly decreased proliferation, decreased expression of MLL-AF6 target genes, and cell cycle arrest of MLL-AF6–transformed cells. These results indicate that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation.

MLL-AF6 Mediated Transformation Is Dependent On the H3K79 Methyl-transferase Dot1l

AbstractAbstract 3502The t(6;11)(q27;q23) produces a chimeric MLL-AF6 oncogene, and is a recurrent chromosomal rearrangement observed in patients with diverse hematologic malignancies such as acute myelogenous leukemia (AML), as well as both B-cell and T-cell acute lymphoblastic leukemias (ALL). The presence of an MLL-AF6 translocation predicts a particularly poor prognosis. Of particular biological interest, the MLL-AF6 translocation is the most common fusion event in which MLL fuses to a predominantly cytoplasmic protein. Very little is known about the molecular mechanisms of transformation mediated by the MLL-AF6 fusion oncogene, forestalling the development of specific therapeutic strategies for t(6;11)(q27;q23) positive leukemias. Recent studies suggest that the histone methyltransferase DOT1L could be an important therapeutic target in MLL-rearranged leukemias. We sought to assess whether MLL-AF6 mediated transformation is also dependent on aberrant H3K79 methylation using genomic, genetic and pharmacological approaches. First, we performed chromatin immuno-precipitation using H3K79me2 specific antibodies followed by next generation sequencing (ChIP-seq) on murine MLL-AF6 leukemias as well as on ML2, the human myelomonocytic leukemia cell line bearing the MLL-AF6 fusion gene. We observed that in both murine and human MLL-AF6 leukemia cells, MLL-fusion target genes display markedly high levels of H3K79 dimethylation as compared to other highly expressed genes. We then investigated whether MLL-AF6-induced transformation was dependent on aberrant H3K79 methylation through genetic or pharmacologic inhibition of the Dot1l histone methyltransferase. Lineage negative/Sca-1 positive/Kit positive (LSK) cells from mice bearing homozygous Dot1l floxed alleles were immortalized by retroviral expression of the MLL-AF6 fusion gene. Cre-recombinase mediated excision of Dot1l from MLL-AF6 transformed bone marrow cells resulted in a significant reduction in H3K79 dimethylation at the promoters of the MLL-target genes Hoxa9, Hoxa10 and Meis1, with a concomitant decrease in their expression. Dot1l excision significantly diminished the clonogenic capacity, abrogated blast colony formation in methylcellulose based medium, and enhanced differentiation of MLL-AF6 transformed cells.We then sought to assess whether EPZ004777, a recently described specific small molecule inhibitor of DOT1L could show efficacy against murine and human MLL-AF6 transformed cells. Dot1l inhibition using EPZ004777 significantly diminished H3K79 dimethylation globally (as assessed by immunoblotting) as well as on MLL-target genes (as assessed by ChIP-qPCR) using H3K79me2 specific antibodies. Importantly, EPZ004777 treatment significantly impaired the proliferation of both murine MLL-AF6 transformed cells as well as the ML2 cell line, whereas the proliferation rates of Hoxa9-Meis1 transformed cells as well as the human MLL-germline cell line HL60 were unaffected despite a similar decrease in H3K79me2 levels. EPZ004777 treatment induced cell cycle arrest as well as increased apoptosis in MLL-AF6 positive, but not control leukemia cells, demonstrating a selective activity of the DOT1L inhibitor EPZ004777 on MLL-AF6 transformed cells.In summary, we demonstrate that the MLL-AF6 oncoprotein requires continued activity of the histone methyltransferase DOT1L for aberrant epigenetic activation of downstream target oncogenes. More studies are needed to understand the mechanisms by which DOT1L is recruited to MLL-target genes by the MLL-AF6 fusion, since AF6 is not believed to normally associate with DOT1L. Nevertheless, the demonstration that H3K79 methylation is important for MLL-AF6 mediated transformation indicates that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation. Disclosures:Olhava:Epizyme: Employment. Daigle:Epizyme, Inc.: Employment. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Armstrong:Epizyme: Consultancy.

Selection for Evi1 activation in myelomonocytic leukemia induced by hyperactive signaling through wild-type NRas

Activation of NRas signaling is frequently found in human myeloid leukemia and can be induced by activating mutations as well as by mutations in receptors or signaling molecules upstream of NRas. To study NRas-induced leukemogenesis, we retrovirally overexpressed wild-type NRas in a murine bone marrow transplantation (BMT) model in C57BL/6J mice. Overexpression of wild-type NRas caused myelomonocytic leukemias ∼3 months after BMT in the majority of mice. A subset of mice (30%) developed malignant histiocytosis similar to mice that received mutationally activated NRas(G12D)-expressing bone marrow. Aberrant Ras signaling was demonstrated in cells expressing mutationally active or wild-type NRas, as increased activation of Erk and Akt was observed in both models. However, more NRas(G12D) were found to be in the activated, GTP-bound state in comparison with wild-type NRas. Consistent with observations reported for primary human myelomonocytic leukemia cells, Stat5 activation was also detected in murine leukemic cells. Furthermore, clonal evolution was detected in NRas wild-type-induced leukemias, including expansion of clones containing activating vector insertions in known oncogenes, such as Evi1 and Prdm16. In vitro cooperation of NRas and Evi1 improved long-term expansion of primary murine bone marrow cells. Evi1-positive cells upregulated Bcl-2 and may, therefore, provide anti-apoptotic signals that collaborate with the NRas-induced proliferative effects. As activation of Evi1 has been shown to coincide with NRAS mutations in human acute myeloid leukemia, our murine model recapitulates crucial events in human leukemogenesis.

Efficient Induction of Extrinsic Cell Death by Dandelion Root Extract in Human Chronic Myelomonocytic Leukemia (CMML) Cells

BackgroundChronic Myelomonocytic Leukemia (CMML) is a heterogeneous disease that is not only hard to diagnose and classify, but is also highly resistant to treatment. Available forms of therapy for this disease have not shown significant effects and patients rapidly develop resistance early on in therapy. These factors lead to the very poor prognosis observed with CMML patients, with median survival duration between 12 and 24 months after diagnosis. This study is therefore centered around evaluating the selective efficacy of a natural extract from dandelion roots, in inducing programmed cell death in aggressive and resistant CMML cell lines.Methodology/Principal FindingsTo confirm the induction of programmed cell death in three human CMML cell lines, nuclear condensation and externalization of the phosphatidylserine, two main characteristics of apoptosis, were detected using Hoechst staining and annexin-V binding assay. The induction of another mode of cell death, autophagy, was determined using a monodansylcadaverine (MDC) stain, to detect the formation of autophagy vacuoles. The results from this study indicate that Dandelion Root Extract (DRE) is able to efficiently and selectively induce apoptosis and autophagy in these cell lines in a dose and time dependent manner, with no significant toxicity on non-cancerous peripheral blood mononuclear cells. More importantly, we observed early activation of initiator caspase-8, which led to mitochondrial destabilization and the induction of autophagy, suggesting that DRE acts through the extrinsic pathway of apoptosis. The inability of DRE to induce apoptosis in dominant-negative FADD cells, confirms the mechanism of action of DRE in in vitro models of CMML.ConclusionThe results from this study indicate that natural products, in particular Dandelion Root Extract, have great potential, as non-toxic and effective alternatives to conventional modes of chemotherapy available today.

Establishment and characterization of a new human acute myelomonocytic leukemia cell line JIH-3

Here, a new acute myelomonocytic leukemia (AMML) cell line, JIH-3, is reported, and its biological characteristics are described. JIH-3 cells were maintained without any cytokines for 27 months. The JIH-3 cell line showed typical myelomonocytic morphological features. Additionally, it mainly expressed myeloid and monocytic markers (CD13, CD14, CD11b, CD15 and CD33), although it also expressed other antigens such as the markers of T and B lymphocytic lineage as well as stem cell, progenitor cell, and natural killer cell-related antigens (CD4, CD5, CD7, CD10, CD22, CD34, CD38, HLADR, CD16/CD56 and CD56); the expression of these markers, suggested that this cell line was in the early stage of myelomonocytic differentiation. Cytogenetic analysis initially showed a karyotype of 46, XY, del(7) (p1?3p2?2). During the passage period, the cells with this karyotype gradually decreased and were replaced by cells with a 45,XY,dic(4;7)(p11;p11),del(15)(q2?2) karyotype. Chromosome painting showed a deletion in the short arm of chromosome 7 for del(7)(p1?3p2?2) and der(4;7)(p11;p11). The latter had larger deleted segment than the former. Fluorescence in situ hybridization (FISH) revealed the dicentric nature of der(4;7), and Multiplex FISH (M-FISH) confirmed that der(4;7) was an unbalanced translocation. A deletion involving the 7p region on dic(4;7)(p11;p11) harbors many genes, including CDC2L5, C7ORF11, C7ORF10 and INHBA. Haploinsufficiency of the genes on 4p, 7p and 15q caused by deletions of 4p, 7p and 15q2?2 that resulted from dic(4;7)(p11;p11) and del(15)(q2?2) may play important roles in leukemogenesis and in the establishment of the JIH-3 cell line. JIH-3 cells did not express multidrug resistance (MDR)-related genes and apoptosis-related genes such as MDR1, multidrug resistance-related protein, lung resistance protein, BCL-2, Bax, GS-π or Fax, only P21 expression was detected, which probably leads the MDR indirectly through inhibition of the activities of cyclin-dependent kinase (CDK). JIH-3 cells had tumorigenic capacity in nude mice. In conclusion, JIH-3 is a new acute myelomonocytic leukemia cell line. It is from a well-characterized background and provides a new useful tool for the study of leukemia patients with a 7p deletion.

NADPH Oxidase Regulates Cytarabine-Induced Apoptotic Death in Acute Myeloid Leukemia Cells

Abstract 4258 Background:Cellular metabolism and oxidative stress are important in the biology and pathophysiology of malignancies. Both increased reactive oxygen species [ROS] levels and induction of anti-oxidative pathways have been described in several malignancies, and may be modulate tumor biology and susceptibility to chemotherapy. Limited studies point to metabolic pathways, including ROS production, influencing pathogenesis and chemo-sensitivity of leukemia. NADPH oxidase is a critical enzyme in antimicrobial host defense and its activation results in ROS generation in myeloid leukemia cells. Our prior studies show that NADPH oxidase can activate Nrf2, a transcriptional factor that induces anti-oxidant and cytoprotective pathways. However the role of NADPH oxidase in chemotherapy-mediated apoptosis induction in leukemic cells is not well-known. Hypothesis:NADPH oxidase-derived ROS will increase sensitivity of AML cells to chemotherapy, whereas Nrf2 will be associated with chemotherapy resistance Methods:We evaluated the role of NADPH oxidase and Nrf2 in regulating cytarabine-induced cell death in wild-type [WT] and engineered PLB-985 cells, a human acute myelomonocytic leukemia cell line derivative. NADPH oxidase-deficient PLB-985 cells were generated by recombination with mutant gp91phox, a necessary component of NADPH oxidase. Nrf2-deficient cells were generated by shRNA (Nrf2shRNA) and depletion (>70%) of Nrf2 mRNA was confirmed by quantitative-PCR. WT and engineered PLB-985 cells were treated with cytarabine (12.5 to 750ng/ml for 24 – 48 hours) and cell death was determined by trypan blue exclusion and Annexin V/7-AAD staining. Results:NADPH oxidase-deficient PLB-985 cells were significantly more resistant to cytarabine compared to WT cells. Cytarabine (500 ng/ml for 48h) induced apoptotic cell death in 25% of NADPH oxidase-deficient vs. 53% of WT PLB-985 cells. Additional dose-response studies confirmed a significant effect of NADPH oxidase in potentiating cytarabine-induced cell death. Nrf2shRNA PLB-985 cells had either similar or modestly increased susceptibility to cytarabine-induced cell death compared to WT PLB-985 cells with empty vectors. NADPH-deficient/Nrf2shRNA PLB-985 cells had similar susceptibility to cytarabine as NADPH-deficient cells with empty vector. Conclusions:Our results show that NADPH oxidase potentiates apoptotic cell death by cytarabine in a myelomonocytic leukemia cell line. However, we did not observe a consistent effect of Nrf2 depletion on apoptotic cell death by cytarabine. These studies suggest that modulation of redox-stress may be a potential therapeutic approach in AML that merits further study. Disclosures:No relevant conflicts of interest to declare.

Isolation of substances with antiproliferative and apoptosis-inducing activities against leukemia cells from the leaves of Zanthoxylum ailanthoides Sieb. & Zucc

Extraction of the leaves of Zanthoxylum ailanthoides Sieb. & Zucc. affords extracts and four isolated compounds which exhibit activities against leukemia cells. The chloroform-soluble fraction (ZAC) of the crude extract of this plant showed cytotoxic activity against human promyelocytic leukemia (HL-60) and myelomonocytic leukemia (WEHI-3) cells with IC(50) values of 73.06 and 42.22 μg/mL, respectively. The active ZAC was further separated to yield pheophorbide-a methyl ester (1), pheophorbide-b methyl ester (2), 13(2)-hydroxyl (13(2)-S) pheophorbide-a methyl ester (3) and 13(2)-hydroxyl (13(2)-R) pheophorbide-b methyl ester (4) whose structures were confirmed by spectroscopic methods. Compounds 2-4 showed cytotoxic activities against both leukemia cells with IC(50) value in the range of 46.76-79.43 nM, whereas compound 1 exhibited only weak cytotoxic activity. The extracts and compounds 1-4 also induced apoptosis and DNA damage in leukemia cells after treatment. The results suggested that the Z. ailanthoides is biologically active against leukemia cells.

The Interferon Consensus Sequence-binding Protein (ICSBP/IRF8) Represses PTPN13 Gene Transcription in Differentiating Myeloid Cells

The interferon consensus sequence-binding protein (ICSBP/IRF8) is an interferon regulatory factor that is expressed in myeloid and B-cells. ICSBP-deficient mice develop a myeloproliferative disorder characterized by cytokine hypersensitivity and apoptosis resistance. To identify ICSBP target genes involved in these effects, we screened a CpG island microarray with chromatin that co-immunoprecipitated with ICSBP from myeloid cells. Using this technique, we identified PTPN13 as an ICSBP target gene. PTPN13 encodes Fas-associated phosphatase 1 (Fap-1), a ubiquitously expressed protein-tyrosine phosphatase. This was of interest because interaction of Fap-1 with Fas results in Fas dephosphorylation and inhibition of Fas-induced apoptosis. In this study, we found that ICSBP influenced Fas-induced apoptosis in a Fap-1-dependent manner. We also found that ICSBP interacted with a cis element in the proximal PTPN13 promoter and repressed transcription. This interaction increased during myeloid differentiation and was regulated by phosphorylation of conserved tyrosine residues in the interferon regulatory factor domain of ICSBP. ICSBP deficiency was present in human myeloid malignancies, including chronic myeloid leukemia. Therefore, these studies identified a mechanism for increased survival of mature myeloid cells in the ICSBP-deficient murine model and in human myeloid malignancies with decreased ICSBP expression.

Flow cytometric measurement of calpain activity in living cells

Calpains are intracellular, calcium-sensitive, neutral cysteine proteases that play crucial roles in many physiological and pathological processes. Calpain regulation is complex and activity is poorly correlated with calpain protein levels. Therefore a full understanding of calpain function requires robust methods for measuring activity. We describe and characterize a flow cytometric method for measuring calpain activity in live cells. This method uses the BOC-LM-CMAC reagent that readily diffuses into cells where it reacts with free thiols to enhance retention. We show that the reagent is cleaved specifically by calpains and follows saturation kinetics. We use the assay to measure calpain activation following PDGF stimulation of rat fibroblasts. We also show that the calpain inhibitor PD150606 inhibits calpain with a K(i) of 12.5 muM and show that Mek inhibitors PD89059 and U0126 also suppress calpain activity. We also show that the assay can measure calpain activity in subpopulations of cells present in unfractionated cord blood or in HL60 human myelomonocytic leukemia cells. Taken together, these experiments demonstrate that this assay is a reliable and useful method for measuring calpain activity in multiple cell types.

Establishment of a Stroma-Dependent Human Acute Myelomonocytic Leukemia Cell Line, NAMO-2, with FLT3 Tandem Duplication

We have established a stroma-dependent myelomonocytic cell line, NAMO-2, with FLT3 internal tandem duplication (FLT3/ITD). Leukemia cells from a patient with acute myelomonocytic leukemia were administered to form subcutaneous tumors in nude mice, which were maintained successively, although we failed to establish continuously growing cells from the original leukemia cell culture. In the cultures of cells from subcutaneous tumors, there were stroma cells that had originated from the nude mice and showed continuous growth. The leukemia cells showed continuous growth dependent on this stroma, and this cell line was named NAMO-2. Detection of FLT3/ITD by the reverse transcriptase polymerase chain reaction (PCR) and genomic PCR showed that NAMO-2 was homozygous for FLT3/ITD. Constitutive activation of FLT3 was detected by Western blotting, and the phosphorylation of Akt, MEK, and STAT5 was also observed. FLT3 kinase inhibitor AG1296 specifically inhibited cell growth. NAMO-2 provides a useful tool to analyze adherence-dependent survival signaling of leukemia with FLT3/ITD and a model for the screening of FLT3 kinase inhibitors.

In vitro Induction of Apoptosis in U937 Cells by Perillyl Alcohol with Sensitization by Pentoxifylline: Increased BCL-2 and BAX Protein Expression

Background: Chemotherapy is effective against a wide variety of tumor cells, although its use is limited by side effects. In vitro experiments and phase I and II trials have shown that phytochemicals such as perillyl alcohol (P-OH) have antitumor effects. Pentoxifylline (PTX), a synthetic methylxanthine used mainly to treat pathologies associated with hematological diseases, sensitizes tumor cells to chemotherapy. The aim of this study was to determine whether PTX amplifies the antitumor effects of P-OH in U937 human myelomonocytic leukemia cells. Methods: Apoptosis was measured by the loss of mitochondrial membrane potential determined by flow cytometry using dihexyloxacarbocyanine iodide (DiOC₆) and propidium iodide. Bcl-2 and Bax protein expression was also assessed by Western blot analysis. Results: P-OH and PTX induced loss of the mitochondrial membrane potential in U937 cells in vitro. Culturing the cells in the presence of both compounds caused a significant increase (p < 0.001) in apoptosis and expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins. However, despite their coexistence, Bax expression prevailed in our experiments. These data suggest that the effects of PTX might be attributable to changes in the mitochondrial membrane potential. Conclusion: PTX sensitizes tumor cells to the anti-neoplastic action of P-OH. These observations may have clinical relevance in the treatment of cancer patients.

Susceptibility of myelomonocytic leukemia U937 cells to the induction of apoptosis by the non-peptidic Bcl-2 ligand HA14-1 is cell cycle phase-dependent

Pharmacological approaches employing agents that bind to the Bcl-2 surface pocket have been used successfully to neutralize the activities of anti-apoptotic Bcl-2 family members, and induce apoptosis. Several reports suggest that Bcl-2 expression/function is cell cycle-dependent. Hence, is killing by Bcl-2 surface pocket binding agents also cell cycle-dependent? In the current study, centrifugal elutriation was used to generate cell cycle phase-enriched preparations of the human myelomonocytic leukemia cell line U937. Elutriated fractions were treated with sub-optimal cytotoxic concentrations of the pro-apoptotic, non-peptidic Bcl-2 pocket-binding agent HA14-1. A concentration of HA14-1 sufficient to kill ∼30–35% of asynchronous cultures minimally affected the progressions of elutriated post-checkpoint G 1, S, G 2/M phase cells, but completely suppressed the progression of pre-G 1 checkpoint G 1 cells. Analyses of trypan blue exclusion, morphology, nuclear condensation, mitochondrial membrane potential, sub-diploid DNA contents, and caspase-3 indicated preferential killing and induction of apoptosis in pre-G 1 checkpoint G 1 and G 2/M phase cells.

An Intact NF-kappaB Pathway is Required for Histone Deacetylase Inhibitor Induced G1 Arrest and Maturation in U937 Human Myeloid Leukemia Cells

The role of NF-kB in regulating G1 arrest in maturation induced by the histone deacetylase inhibitor sodium butyrate (NaB) was examined in human myelomonocytic leukemia cells (U937). Cells stably transfected with an IkB" "super-repressor" lacking phosphorylation sites necessary for proteasomal degradation exhibited diminished IkB" phosphorylation and NF-kB DNA binding upon exposure to TNF-a. When exposed to NaB (1 mM; 48 hr) or 5 nM PMA (24 hr), IkBaM cells displayed a marked reduction in G1 arrest compared to Neo controls. In each case, this was accompanied by a significant reduction in the percentage of cells expressing the differentiation markers CD11a, CD11b, and CD18. The impairment in NaB-induced maturation in mutant cells was associated with a reciprocal increase in apoptosis. In contrast to impairment in NaB- or PMA-induced NF-kB DNA binding, stable expression of the IkBaM did not modify DNA binding of SP1 or AP2 transcription factors. IkBaM mutant cells also displayed impairment in NaB- and PMA-mediated induction of p21CIP1 and phosphorylation (inactivation) of p34cdc2, as well as diminished levels of pRb-bound E2F1. Finally, the NF-kB inhibitor CAPE antagonized NaB- and PMA-related NF-kB DNA binding as well as induction of p21CIP1. Together, these findings suggest that NF-kB plays an important functional role in mediating NaB-induced p21CIP1 induction, G1 arrest, and maturation in human myelomonocytic leukemia cells, and that disruption of the NF-kB pathway causes cells to engage an alternative, apoptotic program.