Absence Of All-trans Retinoic Acid Research Articles

All-Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion.

All-trans retinoic acid (ATRA) promotes myoblast differentiation into myotubes. Leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6) is a candidate ATRA-responsive gene; however, its role in skeletal muscles remains unclear. Here, we demonstrated that during the differentiation of murine C2C12 myoblasts into myotubes, Lgr6 mRNA expression transiently increased before the increase in the expression of the mRNAs encoding myogenic regulatory factors, such as myogenin, myomaker, and myomerger. The loss of LGR6 decreased the differentiation and fusion indices. The exogenous expression of LGR6 up to 3 and 24 h after the induction of differentiation increased and decreased the mRNA levels of myogenin, myomaker, and myomerger, respectively. Lgr6 mRNA was transiently expressed after myogenic differentiation in the presence of a retinoic acid receptor α (RARα) agonist and an RARγ agonist in addition to ATRA, but not in the absence of ATRA. Furthermore, a proteasome inhibitor or Znfr3 knockdown increased exogenous LGR6 expression. The loss of LGR6 attenuated the Wnt/β-catenin signaling activity induced by Wnt3a alone or in combination with Wnt3a and R-spondin 2. These results indicate that LGR6 promotes myogenic differentiation and that ATRA is required for the transient expression of LGR6 during differentiation. Furthermore, LGR6 expression appeared to be downregulated by the ubiquitin-proteasome system involving ZNRF3.

Rexinoids Modulate Effector T Cell Expression of Mucosal Homing Markers CCR9 and α4β7 Integrin and Direct Their Migration In Vitro.

Altering T cell trafficking to mucosal regions can enhance immune responses towards pathogenic infections and cancers at these sites, leading to better outcomes. All-trans-retinoic acid (ATRA) promotes T cell migration to mucosal surfaces by inducing transcription of the mucosal-homing receptors CCR9 and α4β7 via binding to retinoic acid receptors (RARs), which heterodimerize with retinoid X receptors (RXRs) to function. However, the unstable nature and toxicity of ATRA limit its use as a widespread treatment modality for mucosal diseases. Therefore, identifying alternatives that could reduce or eliminate the use of ATRA are needed. Rexinoids are synthetically derived compounds structurally similar to ATRA. Originally named for their ability to bind RXRs, rexinoids can enhance RAR-mediated gene transcription. Furthermore, rexinoids are more stable than ATRA and possess an improved safety profile, making them attractive candidates for use in clinical settings. Here we show that select novel rexinoids act as ATRA mimics, as they cause increased CCR9 and α4β7 expression and enhanced migration to the CCR9 ligand, CCL25 in vitro, even in the absence of ATRA. Conversely, other rexinoids act synergistically with ATRA, as culturing cells with suboptimal doses of both compounds resulted in CCR9 expression and migration to CCL25. Overall, our findings show that rexinoids can be used independently or synergistically with ATRA to promote mucosal homing of T cells in vitro, and lends support for the prospective clinical use of these compounds in immunotherapeutic approaches for pathogenic infections or cancers at mucosal surfaces.

Downregulation of fibronectin 1 attenuates ATRA-induced inhibition of cell migration and invasion in neuroblastoma cells.

Neuroblastoma (NB) is the most common malignant extra cranial solid tumors in children. It has been well established that retinoic acid (RA) inhibits proliferation of neuroblastoma (NB) by blocking cells at G1 phase of the cell cycle. Clinically, RA has been successfully used to treat NB patients. However, the precise mechanism underlying the potent action of RA-treated NB is not fully explored. In this work, we carried out a gene expression profiling by RNA sequencing on all-trans retinoic acid (ATRA)-treated NB cells. Cancer-related pathway enrichment and subsequent protein-protein interaction (PPI) network analysis identified fibronectin 1 (FN1) as one of the central molecules in the network, which was significantly upregulated during ATRA treatment. In addition, we found that although downregulation of FN1 had no significant effects on either cell proliferation or cell cycle distributions in the presence or absence of ATRA, it increased cell migration and invasion in NB cells and partially blocked ATRA-induced inhibition of cell migration and invasion in SY5Y NB cells. Consistent with this finding, FN1 expression levels in NB patients positively correlate with their overall survivals. Taken together, our data suggest that FN1 is a potential target for effective ATRA treatment on NB patients, likely by facilitating ATRA-induced inhibition of cell migration and invasion.

All-trans retinoic acid and human salivary histatin-1 promote the spreading and osteogenic activities of pre-osteoblasts invitro.

Cell‐based bone tissue engineering techniques utilize both osteogenic cells and biomedical materials, and have emerged as a promising approach for large‐volume bone repair. The success of such techniques is highly dependent on cell adhesion, spreading, and osteogenic activities. In this study, we investigated the effect of co‐administration of all‐trans retinoic acid (ATRA) and human salivary peptide histatin‐1 (Hst1) on the spreading and osteogenic activities of pre‐osteoblasts on bio‐inert glass surfaces. Pre‐osteoblasts (MC3T3‐E1 cell line) were seeded onto bio‐inert glass slides in the presence and absence of ATRA and Hst1. Cell spreading was scored by measuring surface areas of cellular filopodia and lamellipodia using a point‐counting method. The distribution of fluorogenic Hst1 within osteogenic cells was also analyzed. Furthermore, specific inhibitors of retinoic acid receptors α, β, and γ, such as ER‐50891, LE‐135, and MM‐11253, were added to identify the involvement of these receptors. Cell metabolic activity, DNA content, and alkaline phosphatase (ALP) activity were assessed to monitor their effects on osteogenic activities. Short‐term (2 h) co‐administration of 10 μm ATRA and Hst1 to pre‐osteoblasts resulted in significantly higher spreading of pre‐osteoblasts compared to ATRA or Hst1 alone. ER‐50891 and LE‐135 both nullified these effects of ATRA. Co‐administration of ATRA and Hst1 was associated with significantly higher metabolic activity, DNA content, and ALP activity than either ATRA or Hst1 alone. In conclusion, co‐administration of Hst1 with ATRA additively stimulated the spreading and osteogenicity of pre‐osteoblasts on bio‐inert glass surfaces in vitro.

ABL1 kinase inhibitors for the treatment of Niemann-Pick disease type A

An attempt has been made to delineate the role of natural and synthetic retinoid receptor ligands on vimentin expression in the human triple-negative breast cancer cells. The effects of currently synthesized triorganotin derivatives of the general formula R3SnX (R is butyl or phenyl, X is isothiocyanate), which are considered RXR ligands, were investigated in the human MDA-MB-231 breast cancer cell line. Studies were evaluated in the presence and absence of all-trans retinoic acid (ATRA), a natural RAR ligand. Vimentin represents the major protein associated with epithelial-mesenchymal transition (EMT), an essential process when the primary tumour transforms into a malignant one. mRNA and proteomic data obtained in this study, based on the PDQuest software protein evaluation and further quantification of proteins by iTRAQ analysis, suggest that vimentin was significantly reduced in the combination of RAR ligand and RXR ligand treatment. Both tested triorganotin compounds showed similarly reduced expression of vimentin, but tributyltin isothiocyanate (TBT-ITC) proved to be more effective than triphenyltin isothiocyanate (TPT-ITC). Furthermore, the effect of natural (9cRA) and synthetic RXR ligands, both chloride and isothiocyanate derivatives, on vimentin expression was compared.

Down-regulation of vimentin by triorganotin isothiocyanates—nuclear retinoid X receptor agonists: A proteomic approach

An attempt has been made to delineate the role of natural and synthetic retinoid receptor ligands on vimentin expression in the human triple-negative breast cancer cells. The effects of currently synthesized triorganotin derivatives of the general formula R3SnX (R is butyl or phenyl, X is isothiocyanate), which are considered RXR ligands, were investigated in the human MDA-MB-231 breast cancer cell line. Studies were evaluated in the presence and absence of all-trans retinoic acid (ATRA), a natural RAR ligand. Vimentin represents the major protein associated with epithelial-mesenchymal transition (EMT), an essential process when the primary tumour transforms into a malignant one. mRNA and proteomic data obtained in this study, based on the PDQuest software protein evaluation and further quantification of proteins by iTRAQ analysis, suggest that vimentin was significantly reduced in the combination of RAR ligand and RXR ligand treatment. Both tested triorganotin compounds showed similarly reduced expression of vimentin, but tributyltin isothiocyanate (TBT-ITC) proved to be more effective than triphenyltin isothiocyanate (TPT-ITC). Furthermore, the effect of natural (9cRA) and synthetic RXR ligands, both chloride and isothiocyanate derivatives, on vimentin expression was compared.

PB1731 ELEVATED FETAL HEMOGLOBIN (HBF) IN ELDERLY AML PATIENTS RECEIVING HYPOMETHYLATING AGENTS: ENHANCEMENT OF HBF INDUCTION BY RETINOIC ACID?

Background:We recently reported elevation of fetal hemoglobin (HbF) as a potential predictor of outcome in elderly AML/MDS patients treated with DNA‐hypomethylating agents (HMA). HbF elevation prior to decitabine (DAC) treatment (Lübbert et al., Br. J. Haematol. 2017; 176:609‐617) as well as HbF induction following 2 courses of DAC treatment (Stomper et al., Haematologica 2019; 104:59‐69) were associated with superior outcome of AML/MDS patients. Interestingly, HbF elevation was shown to be associated with cytogenetic remissions and to be reversed at relapse, suggesting that HbF is preferentially induced in non‐clonal erythroid cells during HMA therapy.Preclinical work suggests that epigenetically active agents such histone deacetylase inhibitors (HDACi) can be combined with retinoic acid to induce HbF.Aims:We now investigated the effect of the add‐on drugs all‐trans retinoic acid (ATRA) and valproic acid (VPA, an HDACi) on HbF induction during DAC treatment in AML patients.Methods:The DECIDER trial addresses the effects of the add‐on of ATRA or VPA to DAC (Grishina et al., BMC Cancer 2015; 15:430). We serially studied HbF levels in 16 AML patients ≥60 years old that received >2 cycles of treatment within the DECIDER trial. Four patients received only DAC, 5 patients DAC+VPA, 5 patients DAC+ATRA, and 2 patients DAC+VPA+ATRA. HbF levels were measured in peripheral blood erythrocytes by HPLC before treatment and sequentially, i.e. after the end of each treatment course. HbF >1.0% was considered induced.Results:In this albeit small cohort, continued treatment appeared to result in variations in maximally attained levels of HbF depending on the type of drug add‐on. As to VPA, the median peak HbF level attained was 1.5% (range, 0.4 ‐ 5.0%) in 9 patients not having received VPA compared to 1.0% (range, 0.2 ‐ 4.8%) in 7 patients having received VPA. The median peak HbF level in the absence of ATRA (9 patients) was 0.8% (range, 0.2 ‐ 2.8%) compared to 2.1% (range, 0.3 ‐ 5.0 %) in 7 patients having received ATRA.Summary/Conclusion:Given that the add‐on of ATRA (but not VPA) to DAC resulted in superior outcome (Lübbert et al., ASH 2016), these preliminary results suggesting a higher induction of HbF in the presence of ATRA warrant prospective, serial HbF determinations in a planned randomized validation trial. Also, RNA‐sequencing studies in AML cell lines treated with DAC ‐/+ ATRA are addressing de‐repression of the gamma‐globin locus in vitro.

Radotinib Induces Apoptosis of CD11b+ Cells Differentiated from Acute Myeloid Leukemia Cells.

Radotinib, developed as a BCR/ABL tyrosine kinase inhibitor (TKI), is approved for the second-line treatment of chronic myeloid leukemia (CML) in South Korea. However, therapeutic effects of radotinib in acute myeloid leukemia (AML) are unknown. In the present study, we demonstrate that radotinib significantly decreases the viability of AML cells in a dose-dependent manner. Kasumi-1 cells were more sensitive to radotinib than NB4, HL60, or THP-1 cell lines. Furthermore, radotinib induced CD11b expression in NB4, THP-1, and Kasumi-1 cells either in presence or absence of all trans-retinoic acid (ATRA). We found that radotinib promoted differentiation and induced CD11b expression in AML cells by downregulating LYN. However, CD11b expression induced by ATRA in HL60 cells was decreased by radotinib through upregulation of LYN. Furthermore, radotinib mainly induced apoptosis of CD11b+ cells in the total population of AML cells. Radotinib also increased apoptosis of CD11b+ HL60 cells when they were differentiated by ATRA/dasatinib treatment. We show that radotinib induced apoptosis via caspase-3 activation and the loss of mitochondrial membrane potential (ΔΨm) in CD11b+ cells differentiated from AML cells. Our results suggest that radotinib may be used as a candidate drug in AML or a chemosensitizer for treatment of AML by other therapeutics.

204: Inducible nitric oxide synthase is upregulated by IL-23/IL-17A axis in inflammatory bowel disease: A study in Algerian patients

Inflammatory bowel diseases (IBDs) are chronic inflammatory diseases of the gastrointestinal tract, which are clinically present as one of two disorders, Crohn’s disease (CD) or ulcerative colitis (UC). The aim of our work was to study the involvement of Th17 subset in bowel disease pathogenesis by nitric oxide (NO) pathway in Algerian patients with IBD. We investigated the correlation between proinflammatory cytokines (IL17, IL23 and IL-6) and NO production in two groups of patients. We analyzed the expression of mRNAs encoding Th17 cytokines, cytokines receptors and NO synthase 2 (NOS2) in plasma of patients. In the same way, the expression of p-STAT3 and NOS2 was measured. We also studied NO modulation by proinflammatory cytokines (IL-17A, IL-6, TNF-α or IL-1β) in presence or absence of All Trans retinoic acid (At RA) in PBMC, monocytes and in colonic mucosa cultures. Analysis of cytokines, cytokines receptors and NOS2 transcripts revealed that levels of mRNA transcripts of the indicated genes are elevated in all IBD groups. Our study shows a significant positive correlation between NO and IL-17A, IL-23, IL-6 levels in plasma of IBD patients. Interestingly, the correlation is significantly higher in patients with active CD. Our study show that both p-STAT3 and iNOS expression were upregulated in PBMCs and colonic mucosa, especially in patients with active Crohn disease.

IL-23/IL-17A Axis Correlates with the Nitric Oxide Pathway in Inflammatory Bowel Disease: Immunomodulatory Effect of Retinoic Acid

Inflammatory bowel diseases (IBDs) are chronic inflammatory diseases of the gastrointestinal tract, which are clinically present as 1 of the 2 disorders, Crohn's disease (CD) or ulcerative colitis (UC) (Rogler 2004). The immune dysregulation in the intestine plays a critical role in the pathogenesis of IBD, involving a wide range of molecules, including cytokines. The aim of this work was to study the involvement of T-helper 17 (Th17) subset in the bowel disease pathogenesis by the nitric oxide (NO) pathway in Algerian patients with IBD. We investigated the correlation between the proinflammatory cytokines [(interleukin (IL)-17, IL-23, and IL-6] and NO production in 2 groups of patients. We analyzed the expression of messenger RNAs (mRNAs) encoding Th17 cytokines, cytokine receptors, and NO synthase 2 (NOS2) in plasma of the patients. In the same way, the expression of p-signal transducer and activator of transcription 3 (STAT3) and NOS2 was measured by immunofluorescence and immunohistochemistry. We also studied NO modulation by proinflammatory cytokines (IL-17A, IL-6, tumor necrosis factor α, or IL-1β) in the presence or absence of all-trans retinoic acid (At RA) in peripheral blood mononuclear cells (PBMCs), monocytes, and in colonic mucosa cultures. Analysis of cytokines, cytokine receptors, and NOS2 transcripts revealed that the levels of mRNA transcripts of the indicated genes are elevated in all IBD groups. Our study shows a significant positive correlation between the NO and IL-17A, IL-23, and IL-6 levels in plasma of the patients with IBD. Interestingly, the correlation is significantly higher in patients with active CD. Our study shows that both p-STAT3 and inducible NOS expression was upregulated in PBMCs and colonic mucosa, especially in patients with active CD. At RA downregulates NO production in the presence of proinflammatory cytokines for the 2 groups of patients. Collectively, our study indicates that the IL-23/IL-17A axis plays a pivotal role in IBD pathogenesis through the NO pathway.

All-Trans Retinoic Acid Enhances the Cytotoxic Effect of T Cells with Anti-CD38 Chimeric Receptor in Acute Myeloid Leukemia

Abstract 1901Majority of AML blasts express CD38, while human hematopoietic stem cells express CD34 but not CD38. Accordingly, we focused on CD38 for a therapeutic target and developed a cellular immunotherapy by using T cells bearing anti-CD38-chimeric antigen receptor (CAR). We recently reported T cells with anti-CD38-CAR efficiently eliminated B-cell lymphoma cells expressing CD38 in vitro and in vivo. However, intensity of CD38 in lymphoma cells is much higher than that in AML cells. Additionally, leukemic stem cells(LSCs) phenotypically express CD34+CD38−. Thus, to fully employ anti-CD38-CAR against AML blasts including LSCs, intensity of CD38 expression need to be raised for the clinical application. All-trans retinoic acid (ATRA) is widely used for treatment of patients with acute promyelocytic leukemia (APL). Interestingly, it has been reported that ATRA enhances CD38 expression on the surface of AML cells. In this study, we investigated whether human peripheral T cells retrovirally transduced with anti-CD38-CAR kills AML cells and furthermore, whether the cytotoxicity is enhanced in the presence of ATRA. First of all, we confirmed that T cells bearing anti-CD38-CAR expressed GFP as well as the anti-goat mouse IgG-PerCP, whose expression is consistent with the expression of anti-CD38-CAR on the T cells. Next, we evaluated the cytotoxic effect of CD38-specific T cells against two AML cell lines (THP-1 and CMK), which highly express CD38 (>99%). Lactate dehydrogenase (LDH) releasing assay and flow cytometry were performed for cytotoxicity. We co-incubated THP-1 or CMK cells with T cells transduced at a variety of effector (E): target (T) ratios for 3 consecutive days in vitro. Interestingly, these assays showed that T cells bearing anti-CD38-CAR were cytotoxic against THP-1 and CMK cells in dose- and time-dependent manners. However, in the cases of KG1a, U937 and HL60 cells which partially express CD38, killing effect was restricted to CD38+ cells by T cells bearing anti-CD38-CAR, and CD38− AML cells remained alive. These results suggested that augmentation of CD38 expression is essential for the sufficient cytotoxicity against AML cells not expressing CD38 by T cells bearing anti-CD38-CAR. We, then, investigated whether ATRA augmented CD38 expression and resultantly enhanced the cytotoxicity against AML cell lines. Interestingly, ATRA augmented CD38 expression in HL60 cells as well as even in KG1a and U937. However, ATRA by itself exerted no effect on cytotoxicity or proliferating activity in KG1, U937 and even in HL60 cells. These results showed that ATRA contributed to enhancement of CD38 expression, but not to cytotoxicity and proliferation on AML cell lines. Next, we attempted to examine cytotoxic effect of T cells with anti-CD38-CAR on KG1, U937 or HL60 cells following ATRA treatment. AML cell lines were co-cultured with the effector T cells transduced for 3 days in the presence of ATRA. The killing effect of T cells bearing anti-CD38-CAR against AML cell lines was limited in the absence of ATRA. Intriguingly, T cells with anti-CD38-CAR exerted enhanced cytotoxic effect on AML cells in the presence of ATRA. Next, we applied our settings above to AML cells freshly isolated from AML patients. Firstly, we confirmed that CD38 expression was enhanced by ATRA in AML cells from patients as observed in AML cell lines. Furthermore, 3-day incubation of patients’ AML cells with T cells bearing anti-CD38-CAR abrogated AML cells in the presence of ATRA. These results indicated that T cells expressing anti-CD38-CAR efficiently eliminated AML cells from patients as well as AML cell line cells through the enhancement of CD38 expression by ATRA. Here, we propose that pre-treatment of ATRA in patient with AML enhances CD38 expression on the leukemic cell surface, which augments the cytotoxic effect of T cells bearing CAR to eradicate leukemic cells including LSCs. Disclosures:No relevant conflicts of interest to declare.

TIS21/BTG2/PC3 enhances downregulation of c-Myc during differentiation of HL-60 cells by activating Erk1/2 and inhibiting Akt in response to all-trans-retinoic acid

Mouse 12-O-tetradecanoyl phorbol-13-acetate inducible sequence 21 (TIS21), an orthologue of human B-cell translocation gene 2 (BTG2) and rat PC3, is a tumour suppressor that belongs to the antiproliferative gene family, and is implicated in a variety of biological processes. c-Myc is a transcription factor and its deregulation is common in leukaemia and lymphomas; the tumours are highly proliferative and often blocked at an earlier phase than the terminal stage of differentiation. The interrelation and the functional interplay of these two different proteins are not defined yet. We have shown here that the tumour suppressor TIS21 negatively regulated c-Myc expression during all-trans-retinoic acid (ATRA)-induced differentiation that accelerated differentiation and reduced proliferation of acute promyelocytic leukaemia (APL) HL-60 cells. TIS21 downregulated c-Myc mRNA and additionally decreased c-Myc protein stability by increasing its phosphorylation at S62 and T58 residues via activation of Erk1/2 and inhibition of PI3K/Akt along with the subsequent activation of GSK-3β in response to ATRA treatment. HL-60 cells treated with GSK-3β or proteosome inhibitors revealed marked accumulation of c-Myc both in the presence and absence of ATRA plus TIS21, confirming ATRA plus TIS21 mediated c-Myc phosphorylation and its consequent degradation in proteosome. Immunoprecipitation assay revealed that TIS21 hindered the interaction of p-Erk1/2 with Akt, thus directly regulating MAPK and Akt activities without interaction with c-Myc. These findings exhibit anticarcinogenic potential of TIS21 via downregulation of c-Myc expression during ATRA induced differentiation of HL-60 cells involving activation and deactivation of two major c-Myc regulators, Erk1/2 and Akt, respectively.

Impact of Protein Phosphatase PP2A on ATRA-Induced Activation of FoxO3a In Acute Promyelocytic Leukemia Cells

AbstractAbstract 2492Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia caused by reciprocal translocations of the long arms of chromosomes 15 and 17, which prevent cellular differentiation into mature neutrophils. The translocation of the promyelocytic leukemia (PML) gene on chromosome 15 and a retinoic acid receptor α (RARα) gene on chromosome 17 generates a PML-RARα fusion protein that inhibits PML-dependent apoptotic pathways in a dominant negative fashion. This fusion protein also blocks granulocytic differentiation by direct transcriptional inhibition of retinoic acid target genes. All-trans retinoic acid (ATRA) arrests cell growth, granulocytic differentiation, and apoptosis in APL cells via proteasome-dependent degradation of PML-RARα fusion protein and subsequent PML-nuclear body (NB) formation. Although PML is the essential component of PML-NBs and functions as a tumor suppressor, disruption of PML-NBs by the PML-RARα fusion protein inhibits endogenous PML tumor-suppressive functions in APL cells. Therefore, degradation of PML-RARα fusion protein and reorganization of PML-NBs during ATRA treatment are regarded as critical cellular responses, similar to the cell growth arrest and apoptosis of leukemia cells. Recently we demonstrated that FoxO3a (also named FKHRL1), a member of the Forkhead family of transcription factors, is a key molecule for the ATRA-induced cellular responses in APL cells (Blood 2010; 115: 3787–3795). In this study, we investigated the mechanism by which FoxO3a is activated by ATRA treatment in a human promyelocytic leukemia cell line NB4. Okadaic acid, a potent PP2A inhibitor, cancelled ATRA-induced dephosphorylation of AKT and its downstream molecule FoxO3a in NB4 cells. Knockdown of endogenous PP2A by siRNA significantly enhanced phosphorylation of both AKT and FoxO3a. These results suggested that PP2A is involved in ATRA-induced dephosphorylation of AKT and FoxO3a. Concomitantly, PP2AC, a catalytic subunit of PP2A, was dephoshorylated at tyrosine 307, and phosphatase activity of PP2A increased after ATRA treatment. Co-immunoprecipitation assay revealed that PP2A constitutively and directly binds to FoxO3a. Using artificial oligopeptides, we demonstrated that enhanced PP2A activity by ATRA directly dephosphorylates phosphothreonine 32 on FoxO3a. In addition, we found that 14-3-3 epsilon binded to phosphorylated FoxO3a in the cytoplasm in the absence of ATRA. After ATRA treatment, however, dephosphorylated FoxO3a dissociated from 14-3-3 epsilon and moved into the nucleus. Confocal microscopic analysis revealed that PP2A-FoxO3a complex partially co-localized with PML-NBs in the nucleus after ATRA treatment. Together, PML orchestrates nuclear networking with PP2A and FoxO3a for ATRA-induced granulocytic differentiation and apoptosis of APL cells. Disclosures:No relevant conflicts of interest to declare.

MicroRNA-150 Is Down-Regulated In Chronic Myeloid Leukemia and MicroRNA-150 Expression Promotes Myeloid Differentiation.

AbstractAbstract 1213MicroRNAs (miRNAs) are key regulators of gene and protein expression and aberrant regulation of transcription factors by miRNAs plays a role in the block in differentiation seen in acute leukemias. We identified differentially expressed miRNAs in chronic myeloid leukemia (CML) using high throughput miRNA profiling of normal and CML samples. Validation by QPCR in 42 CD34 sorted and unsorted patient samples consistently demonstrated decreased or absent miR-150 expression in CML cells as compared to normal cells with expression lowest in myeloid blast crisis (BC) patient samples. In published reports differential miR-150 expression regulates lymphoid development, and increased expression also favors megakaryocytic differentiation over erythroid differentiation via direct regulation of MYB. To determine if decreased or absent miR-150 plays a functional role in CML, we examined 1) if miR-150 expression is Bcr-Abl dependent, 2) if miR-150 expression affects myeloid differentiation, and 3) how miR-150 mediates any detectable phenotype. First, we found that Bcr-Abl represses miR-150 expression in a doxycycline-inducible Bcr-Abl murine cell line; an effect that is reversible with imatinib mesylate treatment. Next, miR-150 was expressed using lentiviral vector transduction in leukemia cell lines and primary CML cells at levels approximating its expression in normal bone marrow. Appropriate controls included an empty vector control and miR-15a and miR-16 vector transduced cells. Decreased cell proliferation and increased myeloid differentiation were observed in 3 acute myeloid leukemia (AML) cell lines with low levels of endogenous miR-150 (HL60, NB4 and PL-21). In HL60 and NB4 cells, miR-150 expressing cells demonstrated significantly increased CD11b expression and morphologic differentiation after exposure to all-trans retinoic acid (ATRA) or 12–0-tetradecanoylphorbol-13-acetate (TPA). Furthermore, increased CD11b expression was observed in the absence of either drug in miR-150 expressing cells. These differences due to miR-150 were not observed in myeloid BC CML cell lines (K562, Kcl-22, or LAMA-84). In CD34 enriched primary cells from two myeloid BC CML patients, however, aberrant miR-150 expression promoted differentiation as demonstrated by increased CD11b or decreased CD34 expression and decreased CFU formation. Lastly, potential miR-150 targets were identified using existing prediction algorithms and microarray gene expression profiling (GEP) of miR150-transduced cell lines or primary BC CML patient cells. GEP data also suggested that miR-150 promotes myeloid differentiation with increased expression of genes associated with monocytic as well as granulocytic differentiation observed in miR-150 cells. MYB expression is increased in CML and it is a validated target of miR-150. Similar to one published study, MYB protein expression was no different in miR-150 over-expressing CML or AML cells. However, MYB protein was decreased in miR-150 over-expressing cells vs. control cells after ATRA exposure suggesting that MYB may target miR-150 in the presence of ATRA. No difference in phenotype or MYB protein was observed in the absence of ATRA in cells transduced with miR-15a and miR-16, which also target MYB. The expression of transcription factors EGR1, EGR2, HES1 and GATA2 was differentially increased in miR-150 expressing cells. ShRNA knockdown of GATA2 in miR-150 cells suggested that increased GATA2 does not contribute to miR-150 induction of CD11b. The transcription factor COUP-TFII (NR2F2), a predicted target of miR-150, interacts with GATA2 to block adipocyte differentiation. MiR-150 expression decreased luciferase activity of a NR2F2 3′UTR luciferase reporter and decreased NR2F2 protein in cell lines. In primary CD34+ CML cells, however, NR2F2 expression is low and likely does not mediate the phenotype seen in miR-150 expressing cells. In conclusion, these data suggest that miR-150 promotes myeloid differentiation, a previously uncharacterized role, and strongly suggest that absent or low miR-150 expression is not simply a lineage marker, but plays a functional role in leukemia cells possibly through loss of miR-150 regulation of MYB and additional currently unidentified targets. Disclosures:Radich:Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Oehler:Pfizer: Research Funding.

Retinoic acid inhibits osteogenic differentiation of mouse embryonic palate mesenchymal cells

All-trans-retinoic acid (ATRA), a known teratogenic factor affecting the development of cleft palate, has been shown to adversely affect craniofacial development. In the present study, we evaluated the effects of ATRA on the osteo-/adipogenic differentiation of mouse embryonic palate mesenchymal (MEPM) cells, which served as a valid model system for investigating the mechanisms regulating osteogenesis during palatogenesis. MEPM cells were derived from gestational day 13 C57BL/6N mouse embryos and induced to differentiate in the presence or absence of ATRA in either osteogenic medium (OM) or control medium (CM). Alkaline phosphatase (ALP) activity assays, von Kossa staining, and RT-PCR assays confirmed that MEPM cells underwent osteogenic differentiation when cultured in OM. Although ATRA induced ALP activity and lipid accumulation in MEPM cells, it failed to induce matrix mineralization and osteoblastic gene expression. BMPR-IB and Smad5 mRNA levels increased significantly in cells cultured in OM and declined following treatment with ATRA, whereas the expression of the BMPR-IA mRNA was up-regulated by ATRA. In conclusion, our results suggested that ATRA and the BMP signaling pathway cooperate to inhibit osteogenesis and promote adipogenesis of MEPM cells.

PML-RARα/RXR Alters the Epigenetic Landscape in Acute Promyelocytic Leukemia

Many different molecular mechanisms have been associated with PML-RARalpha-dependent transformation of hematopoietic progenitors. Here, we identified high confidence PML-RARalpha binding sites in an acute promyelocytic leukemia (APL) cell line and in two APL primary blasts. We found colocalization of PML-RARalpha with RXR to the vast majority of these binding regions. Genome-wide epigenetic studies revealed that treatment with pharmacological doses of all-trans retinoic acid induces changes in H3 acetylation, but not H3K27me3, H3K9me3, or DNA methylation at the PML-RARalpha/RXR binding sites or at nearby target genes. Our results suggest that PML-RARalpha/RXR functions as a local chromatin modulator and that specific recruitment of histone deacetylase activities to genes important for hematopoietic differentiation, RAR signaling, and epigenetic control is crucial to its transforming potential.

Anti-inflammatory effect of retinoic acid on experimental autoimmune uveoretinitis

AimsTo determine whether an active metabolite of vitamin A, all-trans retinoic acid (ATRA), reduces inflammation in experimental autoimmune uveoretinitis (EAU).MethodsNaive CD4+ T cells were activated with anti-CD3, anti-CD28 and transforming...

Differentiating effect of PPARgamma ligand rosiglitazone and all trans-retinoic acid on myeloma cells and its possible mechanism

To investigate the effects of PPARgamma ligand (rosiglitazone, RGZ) as well as combined with all trans-retinoic acid (ATRA) on human myeloma cells and try to explore the possible mechanism. Human myeloma cell lines U266 and RPMI-8226 cells were treated with RGZ in the presence or absence of ATRA. Cell proliferation was evaluated by [(3)H] thymidine incorporation, cell cycle distribution and CD49e expression were analyzed by flow cytometry, morphology changes were evaluated by Wright-Giemsa staining, and p27(Kip1) and p21(Waf1) expression was detected by Western blotting. The exposure to RGZ induced proliferation inhibition in both cell lines in a dose-dependent manner. After cultured with 5 micromol/L RGZ, the proportion of U266 and RPMI-8226 cells in phase G(0)/G(1) was (45.2 +/- 6.7)% and (40.3 +/- 7.3)%, respectively (P < 0.05). The proportion of the cells in phase G(2)/M and S was (52.2 +/- 7.4)% and (57.4 +/- 9.5)%, respectively (P < 0.05). These changes were more evident when the RGZ concentration was increased to 10 micromol/L. A combination of RGZ with ATRA enhanced the growth inhibition and cell cycle arrest effects of RGZ. The RGZ-treated myeloma cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when RGZ was combined with ATRA. These changes were confirmed by the detection of CD49e expression. The expression of p27(Kip1) and p21(Waf1) in myeloma cells was up-regulated by RGZ and this change was more apparent when RGZ was used in combination with ATRA. RGZ can induce cell cycle arrest and cell differentiation in myeloma cells which maybe caused by up-regulation of p27(Kip1) and p21(Waf1) expression. ATRA can enhance these effects of RGZ on multiple myeloma cells and combined use of these two drugs may show a synergistic effect on myeloma cells.

Small Ubiquitin-Like Modifier-2 Modification of Retinoic Acid Receptor-α Regulates Its Subcellular Localization and Transcriptional Activity

The retinoic acid receptor-alpha (Rara) gene is critical for germ cell development in the testis, as demonstrated by infertile Rara knockout male mice. The encoded protein for Rara (RARA) is expressed in both Sertoli cells and germ cells, but it is not always in the nucleus. Previously, all-trans retinoic acid (ATRA) was shown to increase the nuclear localization and transcriptional activity of RARA in Sertoli cells. Here, we identified a small ubiquitin-like modifier-2 (SUMO-2) modification as a novel posttranslational regulatory mechanism controlling the ATRA-dependent RARA subcellular localization and transcription. ATRA increased the SUMO-2 modification of RARA. In the presence of ATRA, lysine 166 (K166) and K171 of RARA were modified at a physiological concentration of SUMO-2, whereas in the absence of ATRA, K399 was the only site that was modified, but at a higher SUMO-2 concentration. However, K399 was critical for ATRA-controlled nuclear trafficking of RARA. In the presence of ATRA, a K399 mutation to arginine resulted in the cytoplasmic localization of K399R mutant, indicating that K166 and K171 sumoylations were inhibitory to nuclear localization. This may be due to SUMO/sentrin-specific peptidase 6 (SENP6) not being able to bind K399R mutant to desumoylate K166 and K171 in Sertoli cells, whereas it can bind RARA with intact K399. On the other hand, functional K166 and K171 sites for sumoylation were required for a full transcriptional activity, when K399 was intact. These results together suggest that both K166 and K171 sumoylation and desumoylation are critical for optimal RARA function.

All-Trans Retinoic Acid Suppresses Stat3 Signaling during Skin Carcinogenesis

Squamous cell carcinoma (SCC) of the skin is the most clinically aggressive form of nonmelanoma skin cancer. We have determined the effects of all-trans retinoic acid (ATRA), a naturally occurring chemopreventive retinoid, on signal transducer and activator of transcription 3 (Stat3) signaling during the development of skin SCC. Stat3 is a transcription factor that plays a critical role in cell proliferation and survival, and it is constitutively active in several malignant cell types. We have previously shown that Stat3 is required for the initiation, promotion, and progression of skin SCC. ATRA is a highly efficient suppressor of tumor formation in the two-stage mouse skin carcinogenesis model and we have shown that this effect correlates with the suppression of the B-Raf/Mek/Erk signaling pathway. In this study, we have determined the pattern of Stat3 phosphorylation throughout the course of the two-stage protocol, both in the presence and absence of ATRA. We have used both SENCAR mice and K5.Stat3C transgenic mice, which express the Stat3C protein, a constitutively active form of Stat3, in the skin. Using Western blotting and immunohistochemical staining with phosphospecific antibodies, we show that coadministration of ATRA suppressed the 12-O-tetradecanoylphorbol-13-acetate-induced phosphorylation of Stat3 in both models, but was only able to suppress tumor formation in the SENCAR mice. Surprisingly, ATRA actually enhanced tumor formation in 12-O-tetradecanoylphorbol-13-acetate-treated K5.Stat3C mice. We hypothesize that ATRA blocks tumor formation, at least in part, by targeting events upstream of Stat3, such as the B-Raf/Mek/Erk pathway, and that in the K5.Stat3C mice, in which Stat3 activity is constitutive, it cannot suppress tumor formation.