A Chimeric Gene, FIP1L1-RARα, Is Isolated from t(4;17)-Positive Acute Promyelocytic Leukemia; a Report of Cloning and Functional Analysis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1820-1820
Author(s):  
Takeshi Kondo ◽  
Akio Mori ◽  
Masahiro Onozawa ◽  
Kaoru Kahata ◽  
Satoshi Hashino ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Luciferase Activity ◽  
Promyelocytic Leukemia ◽  
Leukemia Cells ◽  
Atra Treatment ◽  
Partner Gene ◽  
Vitro Analysis ◽  
In Vitro Analysis

Abstract Acute promyelocytic leukemia (APL) comprises 5 to 10% of acute myelogenous leukemia, the leukemia cells of which is characterized by the differentiation block at the stage of promyelocyte. In APL cells, a reciprocal translocation in chromosome 17 is usually observed, and consequently, a retinoic acid receptor alpha (RARα) gene is rearranged. The rearranged RARα gene is fused to another gene, and as a result, the product of newly composed fusion gene provokes the onset of APL. So far, there are five RARα partner genes reported; PML gene in 15q22, PLZF gene in 11q23, NPM gene in 5q35, NuMA gene in 11q13 Stat5b in 17q21. We recently experienced APL case, in which a chromosomal translocation, t(4;17) was observed. FISH analysis of leukemia cells indicated that RARα gene was translocated. To identify the RARα-partner gene, we performed 5′-rapid amplification of cDNA end (RACE) and identified FIP1L1 gene as RARα-partner gene. It is known that FIP1L1 gene is fused to PGDFRα gene in the hypereosinophilic syndrome. DNA sequence revealed that exon 15 of FIP1L1 gene was fused to exon 3 of RARα gene. In previous reports, five X-RARα have the common character; X-RARα manifests homodimerization. X-RARα suppresses retinoic acid-induced transcription. We examined that FIP1L1-RARα has these two characters. Transient transfection analysis showed that FIP1L1-RARα has the ability to form homodimer. And luciferase assay suggested that FIP1L1-RARα suppressed the retinoic acid-induced transcriptional activity at the physiological concentration of all-trans retinoic acid (ATRA). And the level of luciferase activity suppressed by FIP1L1-RARα was similar to that suppressed by PML-RARα. And the therapeutic concentration of ATRA activated the significant level of luciferase activity. Thus, in vitro analysis suggested that ATRA treatment could be effective to FIP1L1-RARα-positive APL patients. Consistent with the results of in vitro analysis, ATRA treatment was clinically effective, and the patient achieved complete remission after five weeks of ATRA treatment. Collectively, we suggest that FIP1L1-RAR? is the sixth pathogenic gene of APL.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2492-2492
Author(s):  
Yasuhiko Sakoe ◽  
Kumi Sakoe ◽  
Haruo Shimazaki ◽  
Keita Kirito ◽  
Norio Komatsu
Keyword(s):  
Retinoic Acid ◽  
Cell Growth ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Cellular Responses ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Atra Treatment ◽  
Granulocytic Differentiation

Abstract Abstract 2492 Acute 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.

scholarly journals Neutrophil Secondary-Granule Deficiency as a Hallmark of All-Trans Retinoic Acid–Induced Differentiation of Acute Promyelocytic Leukemia Cells

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 803-813 ◽  
Author(s):  
Jun Miyauchi ◽  
Kazuma Ohyashiki ◽  
Yuka Inatomi ◽  
Keisuke Toyama
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Chromatin Condensation ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Leukemia Cells ◽  
Atra Treatment ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Secondary Granule

Acute promyelocytic leukemia (APL) is a neoplasm with the unique chromosomal translocation t(15; 17), which involves the retinoic acid receptor α gene. All-trans retinoic acid (ATRA) has been used for APL patients as a potent therapeutic agent to induce differentiation of leukemia cells. Although polymorphonuclear leukocytes (PMNs) appearing in the blood and bone marrow during ATRA treatment often possess Auer rods, indicating their neoplastic origin, other morphological abnormalities of PMNs have not been elucidated. We studied the morphological changes of APL cells during ATRA treatment at the ultrastructural level. Although most aberrant primary granules, including Auer rods, became morphologically normal in response to ATRA therapy and the nuclei showed chromatin condensation and lobulation, resulting in the emergence of PMNs, the lobulated nuclei often had nuclear filamentous connections and/or nuclear blebs, indicating some pathological process. Furthermore, PMNs, particularly early in ATRA treatment, lacked neutrophil secondary granules as did the PMNs appearing in a culture of APL cells incubated with ATRA, findings consistent with previously reported data that acute myeloid leukemia cell lines do not produce secondary granule proteins even after induction of differentiation towards mature neutrophils. The present data indicate that ATRA is incapable of inducing complete morphological maturation of APL cells and that secondary-granule deficiency may be a hallmark of aberrantly differentiated leukemic cells.

All-Trans-Retinoic Acid (ATRA) Induces Autophagy in Acute Promyelocytic Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2012-2012 ◽  
Author(s):  
Bulent Ozpolat ◽  
Ugur Akar ◽  
Yasuko Kondo ◽  
Gabriel Lopez-Berestein
Keyword(s):  
Cell Death ◽  
Acute Promyelocytic Leukemia ◽  
Terminal Differentiation ◽  
Promyelocytic Leukemia ◽  
Beclin 1 ◽  
Leukemia Cells ◽  
Atra Treatment ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract All-trans-retinoic acid (ATRA), a naturally occurring derivative of vitamin A (retinol), is a potent inducer of cellular differentiation, growth arrest, and apoptosis in various tumor cell lines. ATRA induces terminal differentiation of immature leukemic promyelocytes into normal mature granulocytes in vitro and in vivo, leading to a complete remission in majority of acute promyelocytic leukemia (APL) patients. Thus, this system provides an excellent in vitro model for studying the molecular events taking place during the terminal differentiation of myeloid cells. Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in such physiological processes as the development, homeostasis and elimination of unwanted cells. In contrast to apoptosis, autophagic cell death is caspase-independent and does not involve classic DNA laddering. Recent studies suggest that cancer cells in general tend to undergo less autophagy than their normal counterparts, supporting the contention that defective autophagic cell death plays a role in the process of carcinogenesis. The present study provides the first evidence that ATRA induces autophagy in acute promyelocytic leukemia cells. We demonstrated that treatment of APL (NB4) cells with ATRA resulted in a marked increase in formation of the acidic vesicular organelles demonstrated by acridine orange staining detected by flow cytometry. ATRA treatment caused about 12-fold increase in number of acridine orange positive cells compared to untreated group. Furthermore, ATRA induced marked upregulation of microtubule-associated protein light chain 3 (LC3-II), an important hallmark of autophagy, by western blot analysis. To determine the mechanim by which ATRA induces autophagy we examined expression of Beclin-1, an autophagy-inducing protein, and Bcl-2, an inhibitor of Beclin-1 as well as levels of mTOR, a suppressor of autophagy, upon ATRA treatment. ATRA inhibited Bcl-2, upregulated Beclin-1 expression and reduced induction of activation/phosphorylation of mTOR in NB4 cells. However, rapamycin, a specific inhibitor of mTOR failed to induce autophagy, suggesting that ATRA-mediated autophagy was independent of the mTOR pathway. We are currently testing whether inhibition of Beclin-1 can prevent ATRA-induced autophagy in APL cells. In conclusion, our results revealed that ATRA plays a role in regulation of autophagy by reducing Bcl-2 and inducing protein Beclin-1, which in turn induces autophagy, suggesting a novel action and mechanism of ATRA in regulation of autophagy in APL cells.

scholarly journals Neutrophil Secondary-Granule Deficiency as a Hallmark of All-Trans Retinoic Acid–Induced Differentiation of Acute Promyelocytic Leukemia Cells

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 803-813 ◽  
Author(s):  
Jun Miyauchi ◽  
Kazuma Ohyashiki ◽  
Yuka Inatomi ◽  
Keisuke Toyama
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
Chromatin Condensation ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Leukemia Cells ◽  
Atra Treatment ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Secondary Granule

Abstract Acute promyelocytic leukemia (APL) is a neoplasm with the unique chromosomal translocation t(15; 17), which involves the retinoic acid receptor α gene. All-trans retinoic acid (ATRA) has been used for APL patients as a potent therapeutic agent to induce differentiation of leukemia cells. Although polymorphonuclear leukocytes (PMNs) appearing in the blood and bone marrow during ATRA treatment often possess Auer rods, indicating their neoplastic origin, other morphological abnormalities of PMNs have not been elucidated. We studied the morphological changes of APL cells during ATRA treatment at the ultrastructural level. Although most aberrant primary granules, including Auer rods, became morphologically normal in response to ATRA therapy and the nuclei showed chromatin condensation and lobulation, resulting in the emergence of PMNs, the lobulated nuclei often had nuclear filamentous connections and/or nuclear blebs, indicating some pathological process. Furthermore, PMNs, particularly early in ATRA treatment, lacked neutrophil secondary granules as did the PMNs appearing in a culture of APL cells incubated with ATRA, findings consistent with previously reported data that acute myeloid leukemia cell lines do not produce secondary granule proteins even after induction of differentiation towards mature neutrophils. The present data indicate that ATRA is incapable of inducing complete morphological maturation of APL cells and that secondary-granule deficiency may be a hallmark of aberrantly differentiated leukemic cells.

scholarly journals Fucoidan enhances the therapeutic potential of arsenic trioxide and all-trans retinoic acid in acute promyelocytic leukemia, in vitro and in vivo

Oncotarget ◽  
2016 ◽  
Vol 7 (29) ◽  
pp. 46028-46041 ◽  
Author(s):  
Farzaneh Atashrazm ◽  
Ray M. Lowenthal ◽  
Joanne L. Dickinson ◽  
Adele F. Holloway ◽  
Gregory M. Woods
Keyword(s):  
Retinoic Acid ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Therapeutic Potential ◽  
Promyelocytic Leukemia ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

scholarly journals Characterization of acute promyelocytic leukemia cases with PML-RAR alpha break/fusion sites in PML exon 6: identification of a subgroup with decreased in vitro responsiveness to all-trans retinoic acid

Blood ◽  
1995 ◽  
Vol 86 (4) ◽  
pp. 1540-1547 ◽  
Author(s):  
RE Gallagher ◽  
YP Li ◽  
S Rao ◽  
E Paietta ◽  
J Andersen ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Acute Promyelocytic Leukemia ◽  
High Sensitivity ◽  
Promyelocytic Leukemia ◽  
Reaction Products ◽  
Reading Frame ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Fusion Site

Of 113 acute promyelocytic leukemia cases documented to have diagnostic PML-RAR alpha hybrid mRNA, 10 cases (8.8%) had fusion sites in PML gene exon 6 (V-forms) rather than in the two common hybrid mRNA configurations resulting from breaksites in either PML gene intron 6 (L- forms) or intron 3 (S-forms). In 4 V-form cases, a common break/fusion site was discovered at PML gene nucleotide (nt) 1685, abutting a 3′ cryptic splice donor sequence. The fusion site was proximal to the common site in 1 case and more distal in 5 cases. The open reading frame encoding a PML-RAR alpha gene was consistently preserved, either by an in-frame fusion site or by the insertion of 3 to 127 unidentified nts. In 2 V-form cases, hybridization analysis of the reverse transcriptase-polymerase chain reaction products with a PML-RAR alpha juction probe was required for discrimination from L-form cases. Two V- form subgroups were defined by in vitro sensitivity to all-trans retinoic acid (tRA)-induced differentiation: 4 of 4 cases tested with fusion sites at or 5′ to nt 1685 (subgroup E6S) had reduced sensitivity (EC50 > or = 10(-7) mol/L), whereas 4 of 4 cases with fusion sites at or 3′ to nt 1709 (subgroup E6L) had high sensitivity (EC50 < 10(-8) mol/L) indistinguishable from that of L-form and S-form cases. These results provide the first link between PML-RAR alpha configuration and tRA sensitivity in vitro and support the importance of subclassifying APL cases according to PML-RAR alpha transcript type.

Sign in / Sign up

Export Citation Format

Share Document