Interferon-γ Enhances PML Protein Expression in Acute Promyelocytic Leukemia Cells and Cooperates with All-Trans Retinoic Acid to Induce Maturation of NB4 and MR2 Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5040-5040
Author(s):  
Pengcheng He ◽  
Mei Zhang ◽  
Xiaoning Wang ◽  
Jun Qi ◽  
Jieying Xi ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Line ◽  
Interferon Γ ◽  
Promyelocytic Leukemia ◽  
Nb4 Cells ◽  
Inhibition Effect ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Ifn Γ ◽  
Nbt Reduction

Abstract More than 90% patients with untreated acute promyelocytic leukemia(APL) obtain complete remission clinically by using All-trans retinoic acid(ATRA), a strong differentiation inducer. However, the rapid development of ATRA-resistance brings a new problem to the treatment of APL. Interferon(IFN), as an important cytokine, has broad biological activities. It can not only inhibit the growth of tumor cells, but also reverse the drug-resistance of chemotherapy. As proved by some research, the mechanisms of ATRA-resistance are probably related to lacking some important proteins which synthesized by Interferon-γ (IFN-γ). In order to explore a new way to solve the problem of ATRA-resistance in APL, we investigate the effect and mechanisms of IFN-γ in combination with ATRA on the proliferation/differentiation of NB4 cells(APL cell line with ATRA-sensitiveness) and MR2 cells(APL cell line with ATRA-resistance) respectively. ATRA, IFN-γ and IFN-γ in combination with ATRA were incubated with NB4 and MR2 cells respectively. The cell proliferation was tested by MTT assay, the cell differentiation was tested through light microscope, by NBT reduction test and flow cytometry(FCM). The results showed that ATRA could inhibit the growth of NB4 cells significantly (P<0.05), but it had no effect on the growth of MR2 cells (P>0.05). IFN-γ could inhibit the growth of both NB4 cells and MR2 cells slightly (P<0.05). Moreover, the growth inhibition effect of IFN-γ in combination with ATRA on NB4 and MR2 cells was obviously stronger than that of any single drug group (P<0.05). The results of cell morphology observation, NBT reduction test and CD11b antigen detection showed that ATRA could induce differentiation of NB4 cells significantly (P<0.05), but it had no effect on MR2 cells (P>0.05). Although IFN-γ alone had no effect on the differentiation of either NB4 or MR2 cells (P>0.05), it could augment the differentiation of NB4 cells induced by ATRA (P<0.05) and induce the differentiation of MR2 cells slightly (P<0.05) when it combined with ATRA. Furthermore, we have observed the expression of promyelocytic leukemia(PML) protein by indirect immune fluorescent test. The results showed that the number of fluorescent particles in both NB4 and MR2 cells’ nucleus was increased significantly (P<0.05) when they were incubated with IFN-γ respectively, which indicated IFN-γ could induce the expression of PML protein, a tumor growth inhibitor. It can be seen that IFN-γ could augment the proliferation inhibition effect of ATRA on NB4 and MR2 cells through enhancing the expression of PML protein. Moreover, IFN-γ in combination with ATRA not only can strengthen the induction differentiation effect of ATRA on NB4 cells, but also can partially induce the maturation of MR2 cells with ATRA-resistance.

Proteome Analysis of the Proteins Associated with All-Trans Retinoic Acid Resistance in Acute Promyelocytic Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5042-5042
Author(s):  
Pengcheng He ◽  
Mei Zhang ◽  
Jun Qi ◽  
Xiaoning Wang ◽  
Jieying Xi ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Protein Expression ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Comparative Proteomics ◽  
Promyelocytic Leukemia ◽  
Differentially Expressed ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract Although 90% patients with untreated acute promyelocytic leukemia(APL) obtain complete remission because of the usage of all-trans retinoic acid(ATRA), patients with ATRA-resistance are increased gradually. ATRA-resistance has become one of the main causes which affect the long-term therapeutic efficacy of APL. The mechanisms of ATRA-resistance are complex, which probably involve the metabolism of ATRA, abnormal expression of cellular retinoic acid binding protein(CRABP) and P-glycoprotein(P-gp), mutation of RARα and aberration translocation of APL. However, in these previous researches, it was one or a few proteins but not the entirety proteins that were emphasized on the mechanisms of ATRA-resistance. Comparative proteomics can analyze the entire protein expression in cells in whole and has the superiority in screening the drug-resistance proteins differentially expressed. In order to investigate the mechanisms of ATRA-resistance in APL in whole, we compared and analyzed the protein expression profiles between MR2 cells(APL cell line with ATRA-resistance) and NB4 cells(APL cell line with ATRA-sensitiveness) by comparative proteomics. After the total proteins of MR2 cells and NB4 cells were extracted respectively, they were separated by two-dimensional electrophoresis(2-DE). The differences in proteome profile between MR2 cells and NB4 cells analyzed by ImageMaster™ 2D Platinum software. The average protein spots in 2-DE maps of MR2 and NB4 cells were 1160±51 and 1068±33 respectively. 8 protein spots were selected to be identified by Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS), in which the quantity of the protein differentially expressed was more than two times(≥2 or ≤0.5) between MR2 and NB4 cells’ 2-DE map. They were all successfully identified and their definite information was obtained. Among them, 6 proteins were probably involved in the mechanisms of ATRA-resistance in APL and they were Cofilin-1, Elongation factor 1-beta (EF-1β), Tropomyosin isoform(TM), High mobility group protein B1(HMGB1), Ran-specific GTPase-activating protein (RanGAP1) and Galectin-1. Moreover, so far there was no related report on the roles of HMGB1, RanGAP1 and Galectin-1 in the mechanisms of ATRA-resistance in APL. These differential proteins identified provide the new clues for us to further elucidate the mechanisms of ATRA-resistance from multiple factor.

Aminopeptidase Inhibitor Bestatin Potentiates the Induced-Differentiation Activity of All-trans-Retinoic Acid in NB4 Cells Possibly through Down-Regulating c-myc Expression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4443-4443
Author(s):  
Mao-fang Lin ◽  
Xi-jun Qian
Keyword(s):  
Retinoic Acid ◽  
Tumor Therapy ◽  
Expression Level ◽  
Induced Differentiation ◽  
Cd11b Expression ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Pharmacologic Activity ◽  
Nbt Reduction

Abstract All-trans-retinoic acid (ATRA) represents the sole example of clinically useful cyto-differentiating agent. ATRA treatment alone results in complete remission of nearly 80% patients with acute promyelocytic leukemia (APL). However, the therapeutic use of this compound is limited by a number of problems, including the systemic toxicity and ATRA resistant leukemia. One way to circumvent these problems is to identify the agents capable of enhancing the pharmacologic activity of ATRA. As we know, an aminopeptidase inhibitor, bestatin, had been used as an immunomodulator in anti-tumor therapy. Recently, we have reported bestatin can induce apoptosis in HL-60 and K562 cells. In the present study, we investigated whether bestatin can potentiate the ATRA induced-differentiation of APL cell line NB4 cells and whether changes of transcription factors expression are involved in this course. The cellular morphology observed by optical microscopy, the expression level of CD11b measured by flow cytometry and the nitroblue-tetrazolium (NBT) reduction assay was performed to determine the cyto-differentiation in NB4 cells. The mRNA expression levels of c-myc and c-EBPε in NB4 cells were detected by RT-PCR. NB4 cells incubated with 10nM ATRA plus 100μg/ml bestatin showed more morphologic character of metamyelocyte and band neutrophil than that of the cells treated by ATRA alone. Compared with 10nM ATRA used alone, after treating NB4 cells for 72 hours, the addition of various concentration of bestatin (50μg/ml, 75μg/ml, 100μg/ml) dose-dependently enhancesd NBT reduction of NB4 cells (17.6±2.5 vs. 12.0±2.2, p<0.05; 23.5±3.2 vs. 12.0±2.2, p<0.01; 36.0±8.3 vs. 12.0±2.2, p<0.01, respectively). 100μg/ml bestatin time-dependently increased 10nM ATRA induced NBT reduction of NB4 cells from 24 to 72 hours (p<0.01). The effect of various concentration of ATRA in combination with 100μg/ml bestatin was statistically different with the sum of the effects of individual drugs after subtracting the value of background (31.2±9.1 vs. 12.7±4.3, p<0.01; 39.5±5.0 vs.16.0±1.8, p<0.001; 49.6±5.3 vs. 22.1±1.6, p<0.001, respectively). Moreover, 10nM ATRA plus 100μg/ml bestatin could prominently elevate CD11b expression in NB4 cells compared with ATRA alone treated NB4 cells group(60.58±9.18% vs. 31.95±5.52%, p<0.01), while 100μg/ml bestatin could not induced significant changes in the expression level of CD11b in NB4 cells after 72 hours incubation. The various concentration (50μg/ml, 75μg/ml, 100μg/ml) of bestatin synergizes with 10nM ATRA to down-regulate the expression level of c-myc mRNA (p<0.01), which was inversely correlated with the NBT reduction activity of NB4 cells induced by 10nM ATRA plus various concentration bestatin (r=−0.917, p=0.028). However, 100μg/ml bestatin plus 10nM ATRA could not induce any significant changes in the expression level of c-EBPε mRNA compared with ATRA treated alone group. In conclusion, an aminopeptidase inhibitor bestatin can potentiate ATRA-induced differentiation of NB4 cells, which may be through down-regulating the expression of c-myc in concert with ATRA. Bestatin would be useful in anti-APL therapy by enhancing the pharmacologic activity of ATRA.

DNA vaccination with all-trans retinoic acid treatment induces long-term survival and elicits specific immune responses requiring CD4+ and CD8+ T-cell activation in an acute promyelocytic leukemia mouse model

Blood ◽  
2010 ◽  
Vol 115 (3) ◽  
pp. 653-656 ◽  
Author(s):  
Kouichi Furugaki ◽  
Katerina Pokorna ◽  
Carole Le Pogam ◽  
Masayuki Aoki ◽  
Murielle Reboul ◽  
...  
Keyword(s):  
T Cells ◽  
Retinoic Acid ◽  
Mouse Model ◽  
Immune Responses ◽  
Dna Vaccination ◽  
Interferon Γ ◽  
Promyelocytic Leukemia ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract DNA vaccination and all-trans retinoic acid (ATRA) result in a survival advantage in a mouse model of acute promyelocytic leukemia (APL). Depletion of CD4+ or CD8+ cells abolished this effect. CD4+ depletions of long-term survivors resulted in relapse and death within 3 months, thus demonstrating the need of both CD4+ and CD8+ subsets for the generation of DNA-driven antileukemic immune responses and underscoring a crucial role of CD4+ cells in the maintenance of durable remissions. Degranulation and cytotoxic carboxyfluorescein diacetate succinimidyl ester–based assays showed major histocompatibility complex–restricted APL-specific T cell–mediated immune responses. Sorted APL-specific CD8+CD107a+ T cells showed an increase of antileukemic activity. Effectors from ATRA + DNA–treated mice were shown to secrete interferon-γ when stimulated with either APL cells or peptides from the promyelocytic leukemia-RARα vaccine-derived sequences as detected by ELISpot assays. Our results demonstrate that DNA vaccination with ATRA confers the effective boosting of interferon-γ–producing and cytotoxic T cells in the leukemic mice.

Enhancement of sensitivity by bestatin of acute promyelocytic leukemia NB4 cells to all-trans retinoic acid

2002 ◽  
Vol 26 (12) ◽  
pp. 1097-1103 ◽  
Author(s):  
Takeo Hirano ◽  
Masahiro Kizaki ◽  
Kuniki Kato ◽  
Fuminori Abe ◽  
Natsuko Masuda ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Combined effect of all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia cells in vitro and in vivo

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 264-269 ◽  
Author(s):  
Yongkui Jing ◽  
Long Wang ◽  
Lijuan Xia ◽  
Guo-qiang Chen ◽  
Zhu Chen ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Induced Apoptosis

Abstract All-trans retinoic acid (tRA) and arsenic trioxide (As2O3) induce non–cross-resistant complete clinical remission in patients with acute promyelocytic leukemia with t(15;17) translocation and target PML-RARα, the leukemogenic protein, by different pathways suggesting a possible therapeutic synergism. To evaluate this possibility, this study examined the effect of As2O3 on tRA-induced differentiation and, conversely, the effect of tRA on As2O3-induced apoptosis. As2O3 at subapoptotic concentrations (0.5 μM) decreased tRA-induced differentiation in NB4 cells but synergized with atRA to induce differentiation in tRA-resistant NB4 subclones MR-2 and R4 cells as measured by nitroblue tetrazolium reduction and tRA-inducible genes (TTGII, RARβ, RIG-E). tRA cleaved PML-RARα into distinct fragments in NB4 but not in tRA-resistant MR-2 or R4 cells, whereas As2O3 completely degraded PML-RARα in all 3 cell lines. As2O3-induced apoptosis was decreased by tRA pretreatment of NB4 cells but not of R4 cells and was associated with a strong induction of Bfl-1/A1 expression, a Bcl-2 protein family member. Severe combined immunodeficient mice bearing NB4 cells showed an additive survival effect after sequential treatment, but a toxic effect was observed after simultaneous treatment with tRA and As2O3. These data suggest that combined As2O3 and tRA treatment may be more effective than single agents in tRA-resistant patients. Although in vitro data do not always translate to in vivo response, toxicity and potential drug antagonism may be diminished by decreasing the concentration of As2O3 when given at the same time with therapeutic levels of tRA.

Tissue transglutaminase contributes to the all-trans-retinoic acid–induced differentiation syndrome phenotype in the NB4 model of acute promyelocytic leukemia

Blood ◽  
2010 ◽  
Vol 116 (19) ◽  
pp. 3933-3943 ◽  
Author(s):  
Krisztián Csomós ◽  
István Német ◽  
László Fésüs ◽  
Zoltán Balajthy
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Tissue Transglutaminase ◽  
Promyelocytic Leukemia ◽  
Leukemic Cells ◽  
Neutrophil Granulocytes ◽  
Phagocytic Capacity ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract Treatment of acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA) results in terminal differentiation of leukemic cells toward neutrophil granulocytes. Administration of ATRA leads to massive changes in gene expression, including down-regulation of cell proliferation–related genes and induction of genes involved in immune function. One of the most induced genes in APL NB4 cells is transglutaminase 2 (TG2). RNA interference–mediated stable silencing of TG2 in NB4 cells (TG2-KD NB4) coupled with whole genome microarray analysis revealed that TG2 is involved in the expression of a large number of ATRA-regulated genes. The affected genes participate in granulocyte functions, and their silencing lead to reduced adhesive, migratory, and phagocytic capacity of neutrophils and less superoxide production. The expression of genes related to cell-cycle control also changed, suggesting that TG2 regulates myeloid cell differentiation. CC chemokines CCL2, CCL3, CCL22, CCL24, and cytokines IL1B and IL8 involved in the development of differentiation syndrome are expressed at significantly lower level in TG2-KD NB4 than in wild-type NB4 cells upon ATRA treatment. Based on our results, we propose that reduced expression of TG2 in differentiating APL cells may suppress effector functions of neutrophil granulocytes and attenuate the ATRA-induced inflammatory phenotype of differentiation syndrome.

Curcumin Enhances Differentiation of All-Trans Retinoic Acid (ATRA)-Sensitive and ATRA-Resistant Acute Promyelocytic (APL) Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4456-4456 ◽  
Author(s):  
Ameet R. Kini ◽  
Moolky Nagabhushan ◽  
Martin S. Tallman ◽  
Shantanu Roychowdhury
Keyword(s):  
Retinoic Acid ◽  
Therapeutic Agent ◽  
Atra Treatment ◽  
Differentiation Therapy ◽  
Cd11b Expression ◽  
Nb4 Cells ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Nbt Reduction ◽  
Therapeutic Doses

Abstract The introduction of ATRA-based differentiation therapy has significantly enhanced outcomes in patients with APL. However, retinoic acid syndrome and ATRA-resistance remain significant concerns. It would therefore be useful to develop drugs that reduce the therapeutic doses of ATRA needed, and would be effective in ATRA-resistant cases. We have shown previously that curcumin, the yellow compound isolated from spice turmeric, suppresses the initiation and promotion stages of cancer development. In the present study we evaluated whether curcumin affects differentiation of NB4 APL cells. The NB4 cells were derived from a patient with APL, and differentiate in response to ATRA, while NB4-R1 cells are resistant to ATRA. Treatment of NB4 cells with 5 μM curcumin enhanced ATRA-mediated differentiation. Differentiation was assessed by evaluating CD11b expression, nitroblue tetrazolium (NBT) reduction and by morphologic examination. This curumin-mediated enhanced differentiation was apparent at 1 μM as well as 0.1 μM of ATRA. Curcumin alone did not cause differentiation of the NB4 cells, although higher concentrations of curcumin caused apoptosis. We then examined the effect of curcumin on the ATRA-resistant NB-R1 cells. Addition of ATRA and curcumin together induced differentiation of the NB4-R1 cells, whereas either agent alone did not cause differentiation. The differentiation was characterized by increased CD11b expression, NBT reduction and the typical morphologic changes. In addition, differentiation of the NB4-R1 cells was accompanied by restoration of the PML-oncogenic domains (PODs). These results indicate that curcumin may be another unconventional therapeutic agent in APL, following the successful use of ATRA and arsenic trioxide.

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