scholarly journals Establishment of a human acute promyelocytic leukemia-ascites model in SCID mice

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3404-3409 ◽  
Author(s):  
SY Zhang ◽  
J Zhu ◽  
GQ Chen ◽  
XX Du ◽  
LJ Lu ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Fusion Gene ◽  
Promyelocytic Leukemia ◽  
Scid Mice ◽  
Survival Times ◽  
Cd11b Expression ◽  
Nb4 Cells ◽  
Molecular Features ◽  
Nbt Reduction

Acute promyelocytic leukemia (APL) is an interesting model for cancer research because of the presence of the specific PML-RARalpha fusion gene associated with the clinical response to retinoic acid differentiation therapy. To better understand and improve differentiation induction with retinoic acid, we have established a human APL-ascites model in SCID mice using the NB4 human APL cell line. NB4 (1 x 10(6) cells) were transplanted into the peritoneum (IP) of SCID mice for 1 month. NB4 ascites cells (A-NB4) appeared, which were then engrafted in SCID mice periodically for 18 passages at an interval of 3 to 4 weeks with a 100% success rate of tumor induction. The mean survival times of SCID mice transplanted with 1 x 10(6) A-NB4 cells was 21.6 +/- 2.3 days. Analysis of the biologic characteristics of ninth passage NB4 ascitic cells was performed and they were found to have the morphologic, immunologic, cytogenetic, and molecular features of cultured NB4 cells. Furthermore, A-NB4 cells were capable of differentiating when treated with all-trans retinoic acid (ATRA), as manifested by enhanced NBT reduction and CD11b expression. In vivo treatment with ATRA in SCID mice for 4 days also increased NBT reduction by A-NB4 cells. ATRA treatment significantly prolonged survival time in the group after transplantation (28.1 +/- 6.8 to 29.1 +/- 8.4 days) compared with the control (P < .001). Furthermore, treatment with adriamycin, an effective chemotherapeutic drug in APL, had a strong growth suppressive effect on A-NB4 cells. These results demonstrate that this SCID-APL (NB4 ascites cells) model is a useful preclinical system for evaluating new or known drugs in the treatment of APL.

Synergic effects of arsenic trioxide and cAMP during acute promyelocytic leukemia cell maturation subtends a novel signaling cross-talk

Blood ◽  
2002 ◽  
Vol 99 (3) ◽  
pp. 1014-1022 ◽  
Author(s):  
Qi Zhu ◽  
Ji-Wang Zhang ◽  
Hai-Qing Zhu ◽  
Yu-Lei Shen ◽  
Maria Flexor ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Cross Talk ◽  
Fusion Gene ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Dependent Manner ◽  
Induced Differentiation

Abstract Acute promyelocytic leukemia (APL) is characterized by the specific chromosome translocation t(15;17) with promyelocytic leukemia-retinoic acid receptor-α (PML-RARA) fusion gene and the ability to undergo terminal differentiation as an effect of all-trans retinoic acid (ATRA). Recently, arsenic trioxide (As2O3) has been identified as an alternative therapy in patients with both ATRA-sensitive and ATRA-resistant APL. At the cellular level, As2O3 triggers apoptosis and a partial differentiation of APL cells in a dose-dependent manner; both effects are observed in vivo among patients with APL and APL animal models. To further explore the mechanism of As2O3-induced differentiation, the combined effects of arsenic and a number of other differentiation inducers on APL cell lines (NB4 and NB4-R1) and some fresh APL cells were examined. The data show that a strong synergy exists between a low concentration of As2O3 (0.25 μM) and the cyclic adenosine monophosphate (cAMP) analogue, 8-CPT-cAMP, in fully inducing differentiation of NB4, NB4-R1, and fresh APL cells. Furthermore, cAMP facilitated the degradation of As2O3-mediated fusion protein PML-RARα, a process considered to play a key role in overcoming the differentiation arrest of APL cells. On the other hand, cAMP could significantly inhibit cell growth by modulating several major players in G1/S transition regulation. Interestingly, H89, an antagonist of protein kinase A, could block the differentiation-inducing effect of As2O3potentiated by cAMP. These results thus support the existence of a novel signaling cross-talk for APL maturation, which may deepen understanding of As2O3-induced differentiation in vivo, and thus furnish insights for new therapeutic strategies.

scholarly journals PLZF-RARα, NPM1-RARα, and Other Acute Promyelocytic Leukemia Variants: The PETHEMA Registry Experience and Systematic Literature Review

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1313 ◽  
Author(s):  
Marta Sobas ◽  
Maria Carme Talarn-Forcadell ◽  
David Martínez-Cuadrón ◽  
Lourdes Escoda ◽  
María J. García-Pérez ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Complete Remission ◽  
Acute Promyelocytic Leukemia ◽  
Fusion Gene ◽  
Retinoic Acid Receptor ◽  
Promyelocytic Leukemia ◽  
High Relapse Rate ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Shed Light

It has been suggested that 1–2% of acute promyelocytic leukemia (APL) patients present variant rearrangements of retinoic acid receptor alpha (RARα) fusion gene, with the promyelocytic leukaemia zinc finger (PLZF)/RARα being the most frequent. Resistance to all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) has been suggested in PLZF/RARα and other variant APLs. Herein, we analyze the incidence, characteristics, and outcomes of variant APLs reported to the multinational PETHEMA (Programa para el Tratamiento de Hemopatias Malignas) registry, and we perform a systematic review in order to shed light on strategies to improve management of these extremely rare diseases. Of 2895 patients with genetically confirmed APL in the PETHEMA registry, 11 had variant APL (0.4%) (9 PLZF-RARα and 2 NPM1-RARα), 9 were men, with median age of 44.6 years (3 months to 76 years), median leucocytes (WBC) 16.8 × 109/L, and frequent coagulopathy. Eight patients were treated with ATRA plus chemotherapy-based regimens, and 3 with chemotherapy-based. As compared to previous reports, complete remission and survival was slightly better in our cohort, with 73% complete remission (CR) and 73% survival despite a high relapse rate (43%). After analyzing our series and performing a comprehensive and critical review of the literature, strong recommendations on appropriate management of variant APL are not possible due to the low number and heterogeneity of patients reported so far.

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.

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.

Design and Synthesis of Novel Derivatives of ATRA Demonstrate the Combined Importance of Acid Moiety and Conjugated Double Bonds in Its Binding to PMLRAR-Alpha Oncogene in Acute Promyelocytic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5036-5036 ◽  
Author(s):  
Swati Goel ◽  
Carolina Schinke ◽  
Tushar Bhagat ◽  
Yongkai Mo ◽  
Li Zhou ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Transcriptional Repression ◽  
Leukemic Cell ◽  
Therapeutic Index ◽  
Gene Induction ◽  
Promyelocytic Leukemia ◽  
Double Bonds ◽  
Acid Moiety ◽  
Cd11b Expression

Abstract All trans retinoic acid (ATRA) is the treatment of choice for Acute Promyelocytic Leukemia (APL) and binds to the transcriptional repressor PML-RAR-alpha oncogene. The binding of ATRA to RAR-alpha relieves transcriptional repression and leads to induction of genes important for myeloid differentiation. ATRA consists of an acid moiety at position 15 and is also characterized by conjugated double alkene bonds (Fig1A). The exact contributions of these structural components of ATRA in its clinically important binding to RAR-alpha receptor are not very well studied. In an effort to improve the therapeutic index and to study its binding mechanism, we synthesized novel retinoic acid analogs (retinoids). Our objective was to construct retinoids to test these structure functional correlates. 3 novel retinoids created were synthesized as follows: Compound 13a (Fig1B): consisting of modifying conjugated alkene backbone while keeping acid moiety intact. Compound 13b (Fig1C): consisting of modifying conjugated alkene backbone and converting acid to ester moiety. Compound 13c (Fig1D): consisting of modifying conjugated alkene and cycloalkene ring backbone and keeping acid moiety intact. We tested the ability of these newer retinoids to bind to RAR-alpha as well as inhibit the proliferation of PML-RAR-alpha containing acute promyelocytic leukemic cell line NB4. Reporter assays using a RAR- luciferase demonstrated that Compound 13a successfully relieved transcriptional repression by RAR-alpha and led to downstream gene induction. This was significantly less compared to wild type ATRA demonstrating the importance of C9–C10 double bonds in this binding. Compounds 13b and 13c could not relieve RAR mediated repression of gene induction demonstrating that the acid moiety plays a important role in this binding. Functional studies were consistent with these observations and showed that both ATRA and 13a were able to inhibit the proliferation of APL cells while 13b, 13c were not. Flow cytometry showed that 13a led to partial differentiation of NB4 cells, as evident from CD11b expression after five days of culture. 13b and 13c did not lead to any differentiation, while ATRA led to the greatest expression of CD11b. Our results demonstrate that both the acid moiety and conjugated double bonds are important in binding to RAR-alpha and the subsequent transcriptional repression by retinoic acid. These insights will be important in future efforts to improve the stability and efficacy of ATRA and also have implication in efforts focused on treating ATRA resistant cases of APL. Figure Figure

PML/RARa Represses Transactivation of PSMB10 Via a PU.1-Dependent Manner In Acute Promyelocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3866-3866
Author(s):  
Xianwen Yang ◽  
Ping Wang ◽  
Xujie Zhao ◽  
Huahua Zhu ◽  
Sai-Juan Chen ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Retinoic Acid ◽  
Immune System ◽  
Acute Promyelocytic Leukemia ◽  
Target Genes ◽  
Promyelocytic Leukemia ◽  
Class I ◽  
Luciferase Reporter ◽  
Atra Treatment ◽  
Nb4 Cells

Abstract Abstract 3866 Immunoproteasome is a special form of proteasome which contains three unique interferonγ (IFNγ) induced catalytic subunits, i.e. PSMB8, PSMB9 and PSMB10. Immunoproteasome plays a pivotal role in generating certain peptide antigens for MHC class I presentation. Dysregulation of the immunoproteasome system may contribute to the pathogenesis of certain types of malignancies, including leukemia. Our previous study has identified the target genes of PML/RARa, the initiating factor of acute promyelocytic leukemia (APL) on the genome-wide scale, and demonstrated that PML/RARa could selectively target PU.1-regulated genes, which is a critical mechanism for the pathogenesis of APL. PSMB10, encoding an important composition of immunoproteasome, is one of the identified target genes which are regulated by PML/RARa in this manner. Here we revealed the detailed transcriptional regulation mechanism of PSMB10 in APL. Chromatin immunoprecipitation (ChIP)-PCR assay showed that PML/RARa and PU.1 could bind to the PSMB10 promoter in APL cells, including patient derived NB4 cells and Zn-treated PR9 cells. Re-ChIP assay further demonstrated that PML/RARa and PU.1 co-existed on the same DNA fragment of the PSMB10 promoter, which provided the possibility that PML/RARa and PU.1 could co-regulate the PSMB10 promoter. Using a transient luciferase reporter system, we found that PU.1 transactivated the PSMB10 promoter and PML/RARa repressed the PU.1-dependent transactivation. All-trans retinoic acid (ATRA) could relief the repression caused by PML/RARa. To further demonstrate that the PU.1 site (-37bp∼-29bp) and related retinoic acid response elements (RAREs) (-555bp∼-549bp, -258bp∼-252bp) were essential for PML/RARa to function as an effective repressor, we prepared a series of mutant constructs, including the PU.1-site mutant, the construct mutated on both RARE half (RAREh) sites and two constructs respectively mutated on one of the two RAREh sites, and then transfected them into myeloid U937 cells. From the results of luciferase reporter assays, we found that both PU.1 site and RAREh sites played important roles in PML/RARa-mediated transcriptional repression, moreover, the second RAREh site (-258bp∼-252bp) contributed more than the first one (-555bp∼-549bp). Through electrophoretic mobility shift assay (EMSA), we further determined that PML/RARa could interact with PU.1 through protein-protein interaction, and then bind to the PU.1 site on the PSMB10 promoter. Recent study has shown that ATRA treatment could induce the production of anti-PML/RARa in APL mouse, which implicates that ATRA plays an important role in activating immune system. As the essential elements for immune response, HLA class I antigens (A, B & C) present peptides, which are produced from digested proteins degraded by immunoproteasome, to the surface of antigen-presenting cells. We thus utilized real time RT-PCR to measure the expression of PSMB10 and HLA-A/B/C during ATRA-induced NB4 cells differentiation. We found the levels of PSMB10 and HLA-A/B/C expression were up-regulated in ATRA-treated NB4 cells. These results suggested that the enhanced expression of PSMB10 availed immunoproteasome restoration, which benefited the reactivation of immune system during ATRA treatment therapy. Our results not only demonstrate the detailed transcriptional regulation of PSMB10 in APL but imply the potential function of PSMB10 during ATRA treatment as well. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Retinoic acid is required for and potentiates differentiation of acute promyelocytic leukemia cells by nonretinoid agents

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2122-2129 ◽  
Author(s):  
A Chen ◽  
JD Licht ◽  
Y Wu ◽  
N Hellinger ◽  
W Scher ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Drug Exposure ◽  
Primary Cultures ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Granulocytic Differentiation ◽  
Nb4 Cells

Abstract Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

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