Human Acute Promyelocytic Leukemia Cells Research Articles

Establishment of two-dimensional gel electrophoresis profiles of the human acute promyelocytic leukemia cell line NB4

To explore optimum conditions for establishing a two‑dimensional gel electrophoresis (2-DE) map of the human acute promyelocytic leukemia (APL) cell line NB4 and to analyze its protein profiles, we extracted total proteins from NB4 cells using cell disruption, liquid nitrogen freeze-thawing and fracturing by ultrasound, and quantified the extracted protein samples using Bradford's method. 2-DE was applied to separate the proteins, which were silver-stained in the gel. Well‑separated protein spots were selected from the gel using the ImageMaster™ 2D Platinum analysis system. Moreover, the effects of various protein sample sizes (140, 160 and 180µg) on the 2-DE maps of the NB4 cells were determined and compared. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS), peptide mass fingerprinting (PMF) and database searching were used to identify the proteins. When the quantity of loading proteins was 160µg, clear, well-resolved, reproducible 2-DE proteomic profiles of the NB4 cells were obtained. The average number of protein spots in 3 gels was 1160±51 with an average matching rate of 81%. A total of 10 proteins were identified by mass spectrometry and database queries, certain proteins were products of oncogenes and others were involved in cell cycle regulation and signal transduction. In summary, 2-DE profiles of the proteome of NB4 cells were established and certain proteins were identified by MALDI-TOF-MS and PMF which lay the foundation of further proteomic research of NB4 cells. These data should be useful for establishing a human APL proteome database.

Abstract 4965: Oncoprotein PMLRARα directly suppresses Fas-mediated apoptosis through forming an apoptotic inhibitory complex with c-FLIP in vivo

Abstract Objective: Many genotoxic therapies, including radiation, depend on intact Fas signaling to eradicate cancer cells. Defective Fas signaling is an important cause of cancer resistance to therapy. Restoring Fas apoptosis or sensitizing cancer cells to Fas-mediated apoptosis would improve the efficacy of many cancer therapies. To elucidate a role for specific regulators of Fas signaling in cancer cells, we sought to identify potential modulators of Fas expressed in cancers and target them to selectively sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. Methods: Liquid chromatography tandem mass spectrometry was used to identify Fas-associated proteins; co-immunoprecipitation and Western blot were used to detect interactions of PMLRARα, PML, c-FLIP and Fas, and to examine the components of death-inducing signaling complex (DISC) and caspase-8 cleavage. Deletional mutagenesis was used to map the interaction domains. PML shRNA lentivirus and As2O3 were used to knock down PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis. Mice were transfected with PMLRARα, monitored for survival, and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL. Results: We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase-8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P<.001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Livers from PMLRARα-transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with control vector-transfected mice. Conclusions: PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. These data suggest that PMLRARα is a cancer specific Fas-binding inhibitor of Fas-mediated apoptosis and thus, can contribute to cancer development and resistance to therapy. The newly discovered PMLRARα-Fas and PML-Fas complexes can be sites for modulation of apoptosis. By neutralizing the effect of death receptor inhibitors, such as PMLRARα, we can improve responses to many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4965. doi:1538-7445.AM2012-4965

Abstract 4909: Calcitriol and arsenic trioxide synergistically inhibited proliferation of HL-60 cell line mainly through apoptosis pathway

Abstract Arsenic trioxide (As2O3) has shown substantial efficacy in the treatment of patients with acute promyelocytic leukemia (APL). However, since not all patients could achieve complete remission after As2O3 treatment, it is necessary to investigate a novel treatment method overcoming the resistance of current treatment. However, the therapeutic role of calcitriol against APL has not yet been fully elucidated. In our study, we investigated the anti-leukemic effect of calcitriol in combination with As2O3 on the human APL cell line HL-60. Inhibition of proliferation rate of HL-60 cells was measured by the trypan blue exclusion test was resulted in dose, time- dependent expressions. To evaluate the effects of combination treatment of As2O3 and calcitriol on the cell proliferation, we analyzed the combined effects of As2O3 and calcitriol using CalcuSyn software (BioSoft, Ferguson, MO, USA), which is based on the multiple drug-effect equation of Chou and Talalay (Chou TC, et al. Adv Enzyme Regul 1984): a combination index (CI) of <1, 1, or >1 is defined as synergistic, additive, or antagonistic, respectively. In all combinations, the CI values calculated were less than 1. To investigate the effects of these treatments on the apoptosis pathway, we analyzed the expression of Bax, Caspase-3, Bcl-2 with the Western blot and the RT-PCR. In the Western blot analysis, lower Bcl-2 and higher Bax and Caspase-3 expressions were observed in the As2O3 or calcitriol-treated groups compared to the control group. The combination treatment decreased Bcl-2 expression and increased Bax and Caspase-3 expressions more prominently than the single treatment using either As2O3 or calcitriol. In the RT-PCR analysis, these trends were validated again with the same results. These cells were then labeled with fluorescein-conjugated Annexin V-FITC and propidium iodide (PI), and analyzed with flow cytometry to differentiate whether the main cause of cell death was apoptosis or necrosis. To resultsze, calcitriol in combination with As2O3 synergistically inhibited proliferation of the human APL cell line HL-60. In addition, calcitriol combined with As2O3 more prominently activated the apoptosis pathway than single treatment using either calcitriol or As2O3. The main cause of cell death was also apoptosis. Our results suggest that these novel findings could be applied to improve the therapeutic outcomes of patients with APL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4909. doi:1538-7445.AM2012-4909

Antiproliferative Activity of Flavonoids: Influence of the Sequential Methoxylation State of the Flavonoid Structure

Dracocephalum kotschyi Boiss. has been used as part of an ethnobotanical remedy against many forms of human cancer in Iran. It has been demonstrated that a flavonoid named xanthomicrol from D. kotschyi contributes to its preferential antiproliferative activity against malignant cells. In the present study, the antiproliferative activity of its flavonoid fraction was further characterized. Using liquid-liquid extraction and a semi-preparative reversed-phase HPLC method, eight flavonoid aglycones were isolated from the aerial parts of the plant and their identities were confirmed through MS and NMR analyses as luteolin, naringenin, apigenin, isokaempferide, cirsimaritin, penduletin, xanthomicrol and calycopterin. The in vitro antiproliferative activity of each compound was evaluated against a panel of established normal and malignant cell lines using the MTT assay and some structure-activity relationships were observed. The hydroxyflavones (luteolin, apigenin and isokaempferide) exerted comparable antiproliferative activities against malignant and normal cells, while the methoxylated hydroxyflavones (cirsimaritin, penduletin, xanthomicrol and calycopterin) showed preferential activities against tumor cells. This activity may be of value in treating tumors as it would exert few side effects in normal tissues. Xanthomicrol selectively inhibited the growth of human gastric adenocarcinoma, while calycopterin selectively prevented human acute promyelocytic leukemia and human colon carcinoma cells proliferation.

Activation of Notch Signaling Is An Early Event in the Development of PML-Rara-Induced Acute Promyelocytic Leukemia (APL)

Abstract 2468Acute promyelocytic leukemia (APL, FAB M3) is characterized by the presence of the t(15;17) translocation, resulting in production of the oncogenic transcription factor PML-RARA. We previously reported a gene expression signature of 1121 annotated genes that distinguish APL from the other FAB subtypes and normal promyelocytes (Payton et al, JCI 2009). Within this signature, we identified the Notch ligand JAG1 as being overexpressed in APL samples compared to both other AML subtypes (APL/AML=4.2, FDR<0.5%) and normal promyelocytes (APL/pros=5.66, FDR<0.5%). We then measured JAG1 expression in mouse and cellular models of APL. High levels of JAG1 mRNA and protein were found in both zinc treated PR9 cells (which contain a zinc inducible PML-RARA cassette), and in NB4 cells, a human APL cell line. APL cells derived from the mCG-PML-RARA knockin mouse overexpressed Jag1 mRNA compared to wildtype promyelocytes (APL/pros=10.4, p<0.01). Additionally, Jag1 protein was readily detected in up to 90% of murine APL cells by flow cytometry. Collectively, these results indicate that JAG1 overexpression is a common event in both human and murine APL pathogenesis.Hypothesizing that JAG1 overexpression could lead to Notch signaling in APL cells, we measured Notch signaling in human and murine APL cells. The Notch receptors Notch-1 and Notch-2, the components of the gamma secretase complex, and the transcriptional coactivators of Notch are highly expressed in nearly all human APL samples, suggesting that APL cells are capable of Notch signaling. Using bioinformatic approaches, we found that three previously defined activated Notch signatures were significantly enriched in APL cells (FDR=0.00) and also in PR9 and NB4 cells. Protein levels of cleaved Notch-1 increased in induced PR9 cells with the same kinetics as JAG1 protein, peaking at 16 hours post-induction. In addition, flow cytometry demonstrated the presence of cleaved Notch protein in PR9 and NB4 cells, and both primary and immortalized murine APL samples. To define the consequences of inhibiting Notch signaling in APL cells, we cultured primary murine APL cells in the presence of gamma secretase inhibitors; colony formation in 6/9 tumors was significantly inhibited (p<0.05). Growth of primary murine marrow and spleen cells was unaffected under identical culture conditions.Finally, we investigated the role of Notch signaling in APL pathogenesis in young mCG-PR mice prior to the development of leukemia. Marrow derived progenitors from PML-RARA mice replate serially in methylcellulose cultures, in contrast to wildtype marrow cells, which do not replate. In contrast to primary tumor cells, which were variably dependent on Notch signaling, preleukemic cells were uniformly dependent on Notch signaling for replating activity. In the presence of gamma secretase inhibitors, the replating phenotype is significantly decreased (p<0.01). In contrast, colony formation by wildtype cells is unaffected by inhibition of Notch signaling. Similarly, when Notch signaling in primary marrow cells was genetically inhibited by retroviral transduction of a dominant negative mastermind construct (DNMAML), colony formation by PML-RARA cells was significantly decreased at the first, second and third replatings compared to GFP control cells (p<0.01). Again, wildtype marrow colony formation was unaffected by DNMAML transduction. Importantly, wild type marrow cells transduced with a JAG1 expressing retrovirus did not acquire a replating phenotype, nor did animals transplanted with JAG1 expressing marrow develop leukemia. In conclusion, we have demonstrated overexpression of JAG1 and activation of Notch signaling in human and murine APL cells, and have shown that Notch signaling may play a role in the early pathogenesis of APL. Collectively, these results support a model in which PML-RARA induces the self-renewal of hematopoietic progenitors via a Notch signaling pathway, which may be a critical early step in APL pathogenesis. Disclosures:No relevant conflicts of interest to declare.

Discovery of Critical Functions of Histone Demethylase, PHF8, in Mediating ATRA Response in APL

Abstract 226 Introduction:Transcription deregulation plays a key role in acute leukemogenesis, which is mostly initiated by chimeric transcription factors such as PML–RARa that accounts for almost 100% of acute promyelocytic leukemia (APL). While APL is uniquely sensitive to all trans retinoic acid (ATRA) treatment and has been the paradigm of differentiation/epigenetic therapies, the underlying mechanisms remain largely unknown and are major interests in the field with a potential of extending the success to other hematological malignancies. Previously, others and we have shown that aberrant recruitment of histone deacetylases and polycomb repressive complexes by oligomeric PML-RARa are key for suppression of its downstream targets and promote cellular transformation. ATRA treatment disrupts their bindings and results in de-repression of target genes. However, the identification of the co-activator complex responsible for gene activation upon ATRA treatment remains a major hurdle that significantly hinders the progress of understanding mechanisms underlying the epigenetic therapy. Results and Discussion:Given the critical functions of JmjC-domains containing histone demethylases in mediating transcriptional regulation, we performed a systematic screening for differential interaction between JmjC-histone demethylases and PML–RARa upon ATRA treatment. We identified a highly specific interaction between PML–RARa and Plant Homeodomain Finger 8 (PHF8). To assess its effect on histone methylation, we detected specific reduction of H3K9me2 and enhancement of H3K4me3 in PHF8 transfected cells, consistent with its function as a transcriptional activator. This was further supported by results from chromatin immuno-precipitation (ChIP) assays in human APL cell line (NB4) showing that PHF8 differentially bound and mediated the same histone modifications in RARb promoter and activated its expression. To investigate the functional significance of this interaction, PHF8 was expressed in human NB4 cells harboring PML-RARa or murine primary bone marrow cells transformed by APL fusion proteins. Induction of PHF8 expression significantly decreased their in vitro transformation capacity in the presence of physiological concentrations of ATRA. Conversely, specific down-modulation of PHF8 expression by shRNAs reduced ATRA sensitivity of these cells, suggesting a critical function of PHF8 in mediating ATRA response. We hypothesized that PHF8 may be able to sensitize ATRA resistant cells to the treatment. Hence, we induced expression of PHF8 in the ATRA resistant variant of NB4 line, NB4-MR2. We were able to demonstrate that PHF8 sensitized NB4-MR2 cells to ATRA treatment in vitro. In contrast, NB4-MR2 cells expressing PHF8-F279S, a catalytically inactive mutant could not be sensitized to ATRA treatment, indicating that the enzymatic activity is critical for mediating the ATRA response. Most importantly, NB4-MR2 cells expressing wild type PHF8 were also sensitive to ATRA treatment in vivo and failed to induce leukemia in NOD/SCID mice, which would otherwise succumb to leukemia in a very brief latency.To gain further insights into the molecular regulation of PHF8 in ATRA response, we characterized the potential functions of CDK1-mediated phosphorylation of PHF8. It is known that in ATRA treated leukemic cells Cyclin A translocates into the nucleus where it interacts with CDK1. Activated CDK1 induces phosphorylation of PHF8 at two serine (S33)/ threonine (T84) phosphorylation sites. Our results showed that PHF8 in its phosphorylated form had a much higher binding affinity to PML-RARa. Consistently, ChIP analyses revealed that the binding of PHF8 to the RARb promoter and the resultant activation were significantly augmented when PHF8 was constitutively phosphorylated. Moreover, inhibition of PHF8 dephosphorylation by Okadaic Acid, sensitized NB4-MR2 cells to ATRA treatment. Conclusions:We discovered a novel function of the histone demethylase PHF8 in mediating therapeutic response in both ATRA sensitive and ATRA resistant cells. This function of PHF8 is critically dependent on two phosphorylation sites as well as its histone modification activity. Thus, therapeutic interventions such as phosphatase inhibitors that enhance the PHF8 activity might become useful tools for development of new epigenetic therapies sensitizing leukemic cells to ATRA treatment. Disclosures:No relevant conflicts of interest to declare.

Inhibition of PCGF2 enhances granulocytic differentiation of acute promyelocytic leukemia cell line HL-60 via induction of HOXA7

This study tested the hypothesis that Polycomb Repressive Complex 1 (PRC1) may play a negative role in the granulocytic differentiation of acute promyelocytic leukemia (APL) cells. We first examined the expression of PRC1 genes during all- trans retinoic acid (ATRA)-mediated differentiation of human HL-60 cells, and identified PCGF2 as a gene down-regulated by ATRA in a time-dependent manner. Upon gene silencing of PCGF2 with lentiviral short hairpin RNA, granulocytic differentiation was induced as assessed by differentiation marker gene expression, nitroblue tetrazolium staining, Wright-Giemsa staining, and cell cycle analysis. We next identified HOXA7 as a homeobox gene up-regulated by ATRA and successfully induced granulocytic differentiation by overexpression of HOXA7. We next tested the relationship between PCGF2 and HOXA7 by quantifying the changes in HOXA7 and PCGF2 expression upon PCGF2 gene silencing and HOXA7 overexpression, respectively. HOXA7 expression was up-regulated by PCGF2 gene silencing, while PCGF2 expression remained unchanged by ectopic HOXA7 expression, suggesting PCGF2 as acting upstream of HOXA7. Finally, chromatin immunoprecipitation assay was performed with HOXA7 chromatin. We observed gene-specific reduction in direct binding of Pcgf2 protein to HOXA7 chromatin upon PCGF2 gene silencing. Taken together, these results support the notion that down-regulation of PCGF2 is sufficient to induce granulocytic differentiation of HL-60 cells via de-repression of HOXA7 gene expression. In conclusion, we report that PCGF2, a PRC1 gene, played a negative role in the granulocytic differentiation of human APL cells.

PMLRARα binds to Fas and suppresses Fas-mediated apoptosis through recruiting c-FLIP in vivo

AbstractDefective Fas signaling leads to resistance to various anticancer therapies. Presence of potential inhibitors of Fas which could block Fas signaling can explain cancer cells resistance to apoptosis. We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PML–retinoic acid receptor α (PMLRARα). We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)–derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase 8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P < .001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Taken together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. The presence of PML-Fas complexes across different tissues implicates that PML functions in apoptosis regulation and tumor suppression are mediated by direct interaction with Fas.

Peoniflorin prevents the adhesion between inflammatory endothelial cells and leukocytes through inhibiting the activation of MAPKs and NF‐κB

AbstractThe vascular endothelium can be activated by multiple factors, including lipopolysaccharide (LPS) functioning as a key component of the inflammatory response. Activated endothelium promotes the recruitment of leukocytes mainly by releasing various adhesion molecules and amplifies inflammation via a feedback loop. Peoniflorin, the main active constituent of the roots of Paeonia lactiora Pall., possesses anti‐inammatory, anti‐infective, and anti‐platelet aggregative properties. To elucidate the anti‐inammatory mechanism of peoniflorin, the present study was conducted to address its effects on the adhesion of inflammatory endothelial cells to leukocytes. Peoniflorin substantially reduced adhesion of either human acute monocytic leukemia cells (THP‐1) or human acute promyelocytic leukemia cells (HL‐60) to LPS‐stimulated human umbilical vein endothelial cells (HUVECs). It also markedly down‐regulated the expression of E‐selectin at both the gene and protein levels. However, peoniflorin only slightly reduced expression of intercellular adhesion molecule‐1 (ICAM‐1). Signal pathway analysis indicated that peoniflorin reduced phosphorylation of c‐Jun NH2‐terminal kinase (JNK) and p38 kinase, as well as the phosphorylation and degradation of IκB‐α in HUVECs. These findings suggest that prevention of the adhesion between inflammatory endothelial cells and leukocytes contributes, at least partially, to the anti‐inflammation action of peoniflorin. The anti‐adhesion mechanisms of peoniflorin were involved in the down‐regulation of the activation of mitogen‐activated protein kinases (MAPKs) and nuclear factor‐kappa B (NF‐κB), and a reduction of adhesion molecule expressions in endothelial cells. Drug Dev Res 2010.© 2010 Wiley‐Liss, Inc.

A potent antitumor polysaccharide from the edible brown seaweedHydroclathrus clathratus

AbstractA sulfated polysaccharide H3-a1 [designated polysaccharide fraction, H3-a1, ofHydroclathrus clathratus(HCP)] was isolated and purified from an edible brown seaweedHydroclathrus clathratus; HCP contained about 8.6% protein and a relatively high sulfur content (8.2%). HCP significantly affected the growth of human acute promyelocytic leukemia cells (HL-60), human breast carcinoma (MCF-7) and human hepatocellular carcinoma cancer cell lines. As revealed by flow cytometry, when HCP was co-incubated with HL-60 and MCF-7 cells for 72 h, it induced significant sub-G1 arrest (apoptosis) of these cells. HCP caused G1 phase arrest but did not induce terminal differentiation of these cancer cells.In vivoantitumor tests showed that HCP inhibited tumor growth at doses of 20 and 50 mg kg-1in tumor-bearing BALB/c (supplied by the Laboratory Animal Services Centre of The Chinese University of Hong Kong) mice, but this effect was not due to anin vivoinhibitory effect on tumorigenesis. Nevertheless, HCP suppressed ascitic Sarcoma 180 tumor growth and prolonged the life span of the tumor-bearing mice by about 30–40%. HCP had no observed toxic side effects; it did not affect blood cells, or body, liver or spleen weights of mice 4, 7 and 10 days after treatment. This sulfated polysaccharide induced increased tumor necrosis factor-alpha (TNF-α) level in mouse serum. It also activated peritoneal macrophages to secrete TNF-α and nitric oxide.

423 MULTIMERIN2 effects on tumoural vessel development

Retinoic acid (RA) has a variety of biological effects in mammalian cells and tissues. It is well known that RA induces differentiation of human acute promyelocytic leukemia (APL) HL60 cells, fresh human APL cells, and clinical remission in patients with APL. Retinoylation (acylation of proteins by RA) is a possible pathway for RA action. However, an understanding of the role that retinoylation plays in the actions of RA is lacking. In the current study, several retinoylated proteins were detected in RA-treated HL60 fractions following Mono Q anion exchange chromatography and analysis using two-dimensional polyacrylamide gel electrophoresis. One of the retinoylated proteins was identified as Rho-GDIβ (28 kDa) by TOF-MS and co-migration with Rho-GDIβ (28 kDa). Truncated Rho-GDIβ (23 kDa, N ∆ 19), a product of cleavage by caspase-3, was not retinoylated. RA covalently bound to the Thr2 residue in Rho-GDIβ (5 kDa), which is the second product resulting from the cleavage of Rho-GDIβ (28 kDa) by caspase-3. RA treatment increased the level of Rho-GDIβ (28 kDa) and decreased the level of Rho-GDIβ (23 kDa). RA did not induce caspase-3 activity or Rho-GDIβ mRNA expression. It is likely that retinoylation of Rho-GDIβ increases its metabolic stability.

Rab7b Promotes Megakaryocytic Differentiation of K562 Cells by Activating the Protein Kinase C/Extracellular Signal-Regulated Kinase Dependent Pathway.

Abstract 3612Poster Board III-548Rab proteins are small GTPases belonging to the Ras superfamily and involved in cell protein transportation processes such as internalization and exocytosis. Recently it has been found that several Rab proteins such as Rab7 are also essential for signaling and the controlling of cell proliferation and differentiation. We previously have identified a small GTPase homologous to Rab7, named Rab7b, which is localized to lysosome-associated compartments and is selectively expressed in promyeloid or monocytic cells. We demonstrated that rab7b is involved in monocytic differentiation of human acute promyelocytic leukemia cells. In this study, we detected the expression level of Rab7b during the process of PMA induced megakaryocytic differentiation of K562 cells, transfected Rab7b and its mutants into K562 cells, and investigated the function and signaling mechanism of PMA induced megakaryocytic differentiation of K562 cells. Firstly, we treated K562 cells with PMA for various times, detected the expression level of Rab7b by quantitative PCR and Western blot, and measured the expression level of CD11b, CD14, CD33 and CD41a on the cell surface. Next, we constructed Rab7b, Rab7b-Q67L (active GTPase-deficient mutant), Rab7b-ΔCC (localization deficiency) and Rab7b-RNAi expression vectors, which were then transfected into K562 cells. Megakaryocytic differentiation of cells were analyzed after treated with PMA for various times, including cell morphology and adhesive properties, cell growth arrest, polyploidization, secretion of IL-6 and expression of specific marker CD41a. Because of the considerable role of PKC and ERK1/2 signaling pathways in the megakaryocytic differentiation of K562 cells, we treated various K562 cells with PMA for various times and detected the activation of ERK1/2 by Western blot. Later, cells were inhibited by PKC and ERK1/2 specific inhibitors and treated with PMA for various times, and then the expression of Rab7b and megakaryocytic differentiation of K562 cells were observed. At last, PMA-induced activation of PKC and ERK1/2 signaling has been suggested to regulate cell cycle arrest and megakaryocyte differentiation by modulating the expression of the p21waf/cip1 and the dephosphorylation of the Rb protein, which were then detected by us after transfection of Rab7b and inhibition of PKC/MAPK pathways in K562 cells. The expression of Rab7b was upregulated during the PMA-induced megakaryocytic differentiation of K562 cells. Overexpression of wild-type and active mutant (Q67L) of Rab7b enhanced PMA stimulated megakaryocytic differentiation and ERK1/2 activation. In addition, overexpression of the localization deficient mutant (ΔCC) or RNAi of Rab7b inhibited PMA-stimulated megakaryocytic differentiation and ERK1/2 activation. PMA-induced megakaryocytic differentiation and activation of ERK1/2 in K562 cells was reversed by inhibiting either PKC or ERK1/2 signaling pathway, whereas the mRNA and protein expression of Rab7b was only suppressed in PKC inhibiting groups, indicating that Rab7b mediates a signal for megakaryocytic differentiation downstream of PKC and upstream of ERK1/2. Therefore, we concluded that Rab7b could promote PMA induced differentiation of K562 cells by activating the PKC/ERK1/2 signaling pathway and provide Rab7b as a candidate to connect the PKC pathway and the ERK1/2 pathway in PMA-induced megakaryocytic differentiation of K562 cells. Disclosures:No relevant conflicts of interest to declare.

HL-60 Cell Death Induced by Lycorine Is Due to p21-Mediated Cell Cycle Arrest and TNF-α Signaling Stimulation.

Abstract 4158Lycorine displays various biological functions including remarkable anti-tumor effect. We previously reported that lycorine induced anti-leukemia effect via arresting cell cycle and inducing apoptosis on human acute promyelocytic leukemia (APL) cell line HL-60. To explore the molecular mechanism how lycorine induced HL-60 cell apoptosis, cDNA microarray was used to investigate the expression profile of 494 apoptosis-associated genes. Real-time RT-PCR, Western blotting and immunocytochemistry were used to analyze the expression of related genes, as well as the modification and distribution of related proteins in the presence of lycorine. The results showed that 89 differential genes were expressed significantly (Cy3:Cy5 > 2 or < 0.5) among all the 494 apoptosis-related genes. 78 genes were up-regulated and 11 genes were down-regulated. We are particularly interested in the expression increase of p21 (9.271 folds) and TNF-α (8.242 folds). Furthermore, we found that lycorine could down-regulate p21-related gene expression including Cdc2, Cyclin B, Cdk2 and Cyclin E, promote the truncation of Bid protein and the release of cytochrome c from mitochondria, decrease the phosphorylation of IκB, block the nuclear import of NF-κB and down-regulate expression of survivin. This study revealed that lycorine decreased HL-60 cells survival through p21-mediated cell cycle arrest and stimulation of TNF-α signaling pathway which induced apoptosis. Disclosures:No relevant conflicts of interest to declare.

Antiproliferative Activities of Five Chinese Medicinal Herbs and Active Compounds in Elephantopus scaber

Extracts and fractions prepared from five medicinal herbs used to treat cancer in Chinese folk medicine were tested for their antiproliferative activities on five cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MTT results revealed that the ethanolic extracts possessed stronger antiproliferative activities than the corresponding aqueous ones. The bioactive fractions of the ethanolic extracts were mostly found in n-hexane and/or ethyl acetate fractions, which by TLC analysis were shown to contain terpenes and/or phenolic compounds. The ethyl acetate fraction from the ethanolic extract of Elephantopus scaber showed the highest effect with IC50 values of less than 25 microg/mL on three susceptible cell lines, namely, PC-3 (human prostate carcinoma cell), CNE (human nasopharyngeal carcinoma epithelial cell) and HL-60 (human acute promyelocytic leukemia cell). Further isolation and analysis of this fraction led to the identification of three sesquiterpene lactones, two flavonoids and five triterpenes. MTT results showed that the sesquiterpene lactones were the most effective group with IC50 values ranging from 0.9 to 7.5 microM while the flavonoids possessed moderate to strong effect with IC50 values ranging from 17.3 to 42.6 microM. However, the triterpenes displayed weak effect with IC50 values larger than 50 microM. Among the active compounds, deoxyelephantopin, a sesquiterpene lactone, exhibited the strongest effect on the PC-3, CNE and HL-60 cells, with IC50 values of 4.6, 2.6 and 0.9 microM, respectively. Flow cytometric analysis showed that treatment with deoxyelephantopin caused subG-1 population augmentation in PC-3, CNE and HL-60 cells, suggesting apoptosis was induced in these cells.

A novel vanadyl complex with a polypyridyl DNA intercalator as ligand: A potential anti-protozoa and anti-tumor agent

In the search for new metal-based drugs for the treatment of tumoral and parasitic diseases a vanadyl complex, [V(IV)O(SO(4))(H2O)(2)(dppz)].2H(2)O, that includes the bidentate polypyridyl DNA intercalator dipyrido[3,2-a:2',3'-c]phenazine (dppz), was synthesized, characterized by a combination of techniques, and in vitro evaluated on the human acute promyelocytic leukemia cell line HL-60 and against Dm28c strain epimastigotes of the parasite Trypanosoma cruzi, causative agent of Chagas' disease. EPR spectroscopy suggests a distorted octahedral geometry for the complex with the dppz ligand acting as bidentate, binding through both nitrogen donor atoms in an axial-equatorial mode. An oxo group, two water molecules and a sulphate donor occupy the remainder coordination positions. The complex, as well as the anti-trypanosomal reference drug Nifurtimox, showed IC(50) values in the muM range against T. cruzi Dm28c strain. In addition the complex exhibited excellent in vitro anti-tumor activity against leukemia (HL-60 cell line) comparable to that of cisplatin, inducing cell death by apoptosis with IC(50) values in the micromolar range. Data from gel electrophoresis and atomic force microscopy indicate that the complex interacts with DNA, suggesting that its mechanism of action may include DNA as a target. EPR and (51)V NMR experiments were also carried out with aged aerated solutions of the complex to get insight into the stability of the complex in solution and the species responsible for the in vitro activities observed.

Differential cytotoxic effects of arsenic compounds in human acute promyelocytic leukemia cells

Arsenic trioxide, As 2O 3, has successfully been used to treat acute promyelocytic leukemia (APL). Induction of apoptosis in cancerous cells has been proposed to be the underlying mechanism for the therapeutic efficacy of arsenic. To further understand the cytotoxicity of arsenic compounds in APL cells, HL-60 cells were exposed to graded concentrations of the following arsenicals for up to 48 h: arsenic trioxide (As III), sodium arsenate (As V), phenylarsine oxide (PAO III), monomethylarsonous acid (MMA III), monomethylarsonic acid (MMA V) and dimethylarsinic acid (DMA V), and the viability and modes of cell death assessed. The arsenic-exposed cells were stained with annexin V-PE and 7-aminoactinomycin D (7-AAD) and analyzed by flow cytometry in order to detect apoptotic and viable cells while cell morphology was visualized using scanning and transmission electron microscopy. Acridine orange staining and microtubule-associated protein 1 light chain 3 (MAP-LC3) detection were used to recognize autophagic cell death. The results showed that the compounds reduced viable HL-60 cells by inducing apoptosis in a concentration-dependent manner. None of the compounds tested caused a significant change in binding of acridine orange or redistribution of MAP-LC3. Potencies of the six different arsenic compounds tested were ranked as PAO III > MMA III ≥ As III > As V > MMA V > DMA V. An increase in caspase-3 activity by PAO III, MMA III and DMA V implied that these compounds induced apoptosis in HL-60 cells through a caspase-dependent mechanism, but the other arsenic compounds failed to activate caspase-3, suggesting that they induce apoptosis by an alternative pathway.

The Anti-Apoptotic Gene BCL2A1 Is Transcriptionally Regulated by PU.1

PU.1 is a hematopoietic transcriptional regulator that is necessary for the development of both myeloid and B cells. To identify new PU.1 target genes in neutrophil development PU.1 was introduced into mouse 503 PU.1-null cells using lentiviral gene transfer and microarray analyses of two independent 503 PU.1-rescued and parental 503 cells were compared. The BCL2A1 gene was found to be more than 50-fold induced in 503 PU.1- restored as compared to the parental 503-null cells. BCL2A1 (also known as BFL-1/A1) is an anti-apoptotic member of the BCL2 family. BCL2A1 was initially identified as a tissue-specific BCL2-related factor that is induced by different reagents such as granulocyte macrophage colony-stimulating factor (GM-CSF) or all-trans retinoic acid (ATRA) during myeloid differentiation. Upregulation of BCL2A1 in granulocytes may promote a time-dependent survival. To follow up on our microarray findings we evaluated loss of PU.1 function in human NB4 acute promyelocytic leukemia (APL) cells using lentivector delivered, short hairpin (sh) RNAs targeting PU.1. Knockdown efficacy upon ATRA-treatment in the two shPU.1 expressing NB4 cell lines was 67 and 30%, respectively. Silencing of PU.1 markedly reduced BCL2A1 mRNA induction upon ATRA-treatment from 167-fold in control cells to 47- and 112-fold in the two PU.1 knockdown NB4 cell lines, respectively (Figure A). Co-transfection of PU.1 with a human BCL2A1 promoter reporter resulted in a 7-fold activation, suggesting PU.1 can directly regulate BCL2A1. Co-transfection with NF-kappaB, used as positive control, induced the BCL2A1 promoter 14.5-fold. Moreover, in vivo binding of the transcription factor PU.1 to 2/8 putative PU.1 binding sites in the BCL2A1 promoter was shown by chromatin immunoprecipitation in HL60 promyelocytic cells further supporting a role for PU.1 regulation of BCL2A1. Evaluation of BCL2A1 and PU.1 mRNA expression in CD34+ hematopoietic progenitors, granulocytes, and primary acute myeloid leukemia (AML) cells was assessed using real-time quantitative RT-PCR. BCL2A1 and PU.1 mRNA levels were significantly lower in primary AML patient samples (n=80; p<0.0001) and in CD34+ progenitor cells (n=4; p=0.0095) than in granulocytes (n=6; Figure B). In line with this observation, we found that upon ATRA therapy BCL2A1 levels were increased in 5/5 APL patients and PU.1 mRNA levels in 4/5 APL cases, respectively. Altogether, these results clearly indicate that PU.1 and BCL2A1 are co-regulated during granulocyte differentiation. Lastly, we confirmed earlier data showing that ATRA-pretreatment of NB4 cells and thus induction of PU.1 and BCL2A1, rendered these cells less sensitive to arsenic trioxide (As2O3)- induced cell death. Conversely, NB4 PU.1 knockdown cells were markedly more sensitive to As2O3 -induced cell death upon ATRA-pretreatment than the parental NB4 control cells. The increase in sensitivity to As2O3 correlated with the lower BCL2A1 levels found in the PU.1 knockdown cells. In summary, we identified the anti-apoptotic BCL2A1 gene as direct, transcriptional target of PU.1 in myeloid leukemic cells. We hypothesize that PU.1-dependent induction of BCL2A1 is necessary for the survival of normal, terminally differentiated myeloid cells. Furthermore, aberrant expression of PU.1 in erythroleukemia may result in elevated BCL2A1 levels that support increased survival of erythroblasts in this particular type of leukemia. [Display omitted]

Potential Role of Heme Oxygenase-1 in Arsenic Trioxide and Hemin-Mediated Differentiation of Human Promyelocytic Leukemia Cells

Acute promyelocytic leukemia (APL) is a myeloid-derived leukemia characterized by a block of differentiation at the promyelocytic stage of development. APL is associated with a reciprocal translocation of chromosomes 15 and 17 involving the retinoic acid receptor-alpha (RARα) locus and the promyelocytic leukemia locus (PML) forming a new fusion gene, PML/RARα. The fusion protein prevents the cells from differentiating by repressing transcription of target genes necessary for differentiation. Therapy with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) have improved the clinical outcomes in APL. ATRA differentiates APL cells by promoting degradation of PML/RARα and relieving transcriptional repression. ATO induces apoptosis and differentiation of APL cells through separate pathways involving activation of apoptotic pathways and degradation of the PML/RARα protein. The heme-catabolizing protein heme oxygenase-1 (HO-1) which produces carbon monoxide, biliverdin/bilirubin and releases Fe/ferritin can be cytoprotective and anti-apoptotic. HO-1 message and protein are markedly upregulated in response to hemin. ATO also can induce HO-1 expression and has been shown to induce differentiation of leukemic cells. We hypothesize that induction of HO-1 mediates APL differentiation in response to ATO and hemin. We utilized a human APL cell line, NB4, to test our hypothesis in vitro using NBT reduction to assess cell differentiation. ATRA (p < 0.005 at both 72 and 96 hours), ATO (p < 0.005 at 72 hours, and p < 0.05 at 96 hours), and hemin (p < 0.05 at both 72 and 96 hours, n=3) were shown to differentiate NB4 cells compared to untreated groups. RT-PCR indicated HO-1 expression increased in response to ATRA, ATO, and hemin, while biliverdin reductase (BVR), a signaling protein that degrades biliverdin to bilirubin, was only induced by hemin. Inhibition of HO-1 activity with tin-protoporphyrin significantly (p<0.05, n=2) blocked differentiation induced by ATO but had no effect on ATRA–treated cells. We speculate that HO-1 and its downstream metabolic products play critical roles in ATO-induced differentiation of APL cells.

Isolation, characterization, molecular cloning and modeling of a new lipid transfer protein with antiviral and antiproliferative activities from Narcissus tazetta

A fetuin-binding peptide with a molecular mass of about 9 kDa (designated NTP) was isolated and purified from the bulbs of Chinese daffodil, Narcissus tazetta var. chinensis L., by gel filtration and high-performance liquid chromatography, after removing the mannose-binding proteins by mannose–agarose column. Molecular cloning revealed that NTP contained an open reading frame of 354 bp encoding a polypeptide of 118 amino acids which included a 26-amino-acid signal peptide. An analysis of the deduced amino acid sequence of NTP shows considerable sequence homology to the non-specific lipid transfer proteins (nsLTPs) of certain plants. Model of the three-dimensional (3D) structure of NTP exhibits an internal hydrophobic cavity which can bind lipid-like molecules and transfer a wide range of ligands. As a member of the putative non-specific lipid transfer protein of N. tazetta, NTP did not possess hemagglutinating activity toward rabbit erythrocytes. In a cell-free system, it could arrest the protein synthesis of rabbit reticulocytes. Using the in vitro antiviral assays, NTP could significantly inhibit the plaque formation by respiratory syncytial virus (RSV) and the cytopathic effect induced by influenza A (H1N1) virus, as well as the proliferation of human acute promyelocytic leukemia cells (HL-60).

Acetylation of PML Is Involved in Histone Deacetylase Inhibitor-mediated Apoptosis

PML is a potent tumor suppressor and proapoptotic factor and is functionally regulated by post-translational modifications such as phosphorylation, sumoylation, and ubiquitination. Histone deacetylase (HDAC) inhibitors are a promising class of targeted anticancer agents and induce apoptosis in cancer cells by largely unknown mechanisms. We report here a novel post-transcriptional modification, acetylation, of PML. PML exists as an acetylated protein in HeLa cells, and its acetylation is enhanced by coexpression of p300 or treatment with a HDAC inhibitor, trichostatin A. Increased PML acetylation is associated with increased sumoylation of PML in vitro and in vivo. PML is involved in trichostatin A-induced apoptosis and PML with an acetylation-defective mutation shows an inability to mediate apoptosis, suggesting the importance of PML acetylation. Our work provides new insights into PML regulation by post-translational modification and new information about the therapeutic mechanism of HDAC inhibitors.