Acute promyelocytic leukemia with a PLZF-RAR[alpha ] fusion protein

2001 ◽  
Vol 38 (1) ◽  
pp. 37-41 ◽  
Author(s):  
Joop H. Jansen ◽  
Bob L[ouml ]wenberg
Keyword(s):  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia

scholarly journals The PML/RAR alpha oncoprotein is a direct molecular target of retinoic acid in acute promyelocytic leukemia cells

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2826-2832 ◽  
Author(s):  
JV Raelson ◽  
C Nervi ◽  
A Rosenauer ◽  
L Benedetti ◽  
Y Monczak ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Myeloid Differentiation ◽  
Wild Type ◽  
Retinoid Receptor ◽  
Western Blots

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA-responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells.

scholarly journals Immunocytochemical Diagnosis of Acute Promyelocytic Leukemia (M3) With the Monoclonal Antibody PG-M3 (Anti-PML)

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 4046-4053 ◽  
Author(s):  
Brunangelo Falini ◽  
Leonardo Flenghi ◽  
Marta Fagioli ◽  
Francesco Lo Coco ◽  
Iole Cordone ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Detection System ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Nuclear Staining ◽  
Wild Type ◽  
Amino Terminal ◽  
Pml Nuclear Bodies

Abstract Acute promyelocytic leukemia (APL) is characterized by a reciprocal 15; 17 chromosomal translocation, which fuses the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes, leading to the expression of the PML/RARα fusion oncoprotein. Immunocytochemical labeling of the wild-type PML protein with the PG-M3 monoclonal antibody (MoAb) directed against the amino terminal portion of the human PML gene product, produces a characteristic nuclear speckled pattern that is due to localization of the protein into discrete dots (5 to 20 per nucleus), named PML nuclear bodies. The architecture of PML nuclear bodies appears to be disrupted in APL cells that bear the t(15; 17), thus resulting in a change of the nuclear staining pattern from speckled (wild-type PML protein) to microgranular (PML-RARα fusion protein). To assess whether the PG-M3 MoAb could assist in the diagnosis of APL (M3), bone marrow and/or peripheral blood samples from 100 cases of acute nonlymphoid leukemias of different subtypes were blindly immunostained with the PG-M3 MoAb, using the immunoalkaline phosphatase (APAAP) or immunofluorescence technique as detection system. Notably, the abnormal (micropunctate) pattern of the PML/RARα fusion protein (usually ≥50 small granules/per nucleus) was observed in APL (M3) samples, but not in other types of acute nonlymphoid leukemias. Immunocytochemical labeling with PG-M3 was particularly useful in the diagnosis of microgranular variant of APL (M3V) (three cases misdiagnosed as M4 and M5), and also to exclude a morphologic misdiagnosis of APL (six of 78 cases). In all cases investigated, immunocytochemical results were in agreement with those of reverse transcription-polymerase chain reaction (RT-PCR) for PML/RARα. Because the epitope identified by PG-M3 is located in the aminoterminal portion of PML (AA 37 to 51), the antibody was suitable for recognizing APL cases characterized by breakpoint occurring at different sites of PML (bcr 1, bcr 2 and bcr 3). In conclusion, immunocytochemical labeling with PG-M3 represents a rapid, sensitive, and highly-specific test for the diagnosis of APL that bears the t(15; 17). This should allow an easy and correct diagnosis of this subtype of acute leukemia to any laboratory provided with a minimal equipment for immunocytochemistry work.

scholarly journals In vivo analysis of the role of aberrant histone deacetylase recruitment and RARα blockade in the pathogenesis of acute promyelocytic leukemia

2006 ◽  
Vol 203 (4) ◽  
pp. 821-828 ◽  
Author(s):  
Hiromichi Matsushita ◽  
Pier Paolo Scaglioni ◽  
Mantu Bhaumik ◽  
Eduardo M. Rego ◽  
Lu Fan Cai ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Histone Deacetylases ◽  
Retinoic Acid Receptor ◽  
Promyelocytic Leukemia ◽  
Dominant Negative

The promyelocytic leukemia–retinoic acid receptor α (PML-RARα) protein of acute promyelocytic leukemia (APL) is oncogenic in vivo. It has been hypothesized that the ability of PML-RARα to inhibit RARα function through PML-dependent aberrant recruitment of histone deacetylases (HDACs) and chromatin remodeling is the key initiating event for leukemogenesis. To elucidate the role of HDAC in this process, we have generated HDAC1–RARα fusion proteins and tested their activity and oncogenicity in vitro and in vivo in transgenic mice (TM). In parallel, we studied the in vivo leukemogenic potential of dominant negative (DN) and truncated RARα mutants, as well as that of PML-RARα mutants that are insensitive to retinoic acid. Surprisingly, although HDAC1-RARα did act as a bona fide DN RARα mutant in cellular in vitro and in cell culture, this fusion protein, as well as other DN RARα mutants, did not cause a block in myeloid differentiation in vivo in TM and were not leukemogenic. Comparative analysis of these TM and of TM/PML−/− and p53−/− compound mutants lends support to a model by which the RARα and PML blockade is necessary, but not sufficient, for leukemogenesis and the PML domain of the fusion protein provides unique functions that are required for leukemia initiation.

The Acute Promyelocytic Leukemia-Associated Fusion Protein PML/RAR Blocks t-RA-Induced Differentiation in a Subset of Cells with Stem Cell Potential.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1175-1175
Author(s):  
Xiaomin Zheng ◽  
Anita Seshire ◽  
Elena Puccetti ◽  
Hilal Gul ◽  
Tim Beissert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Fusion Protein ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Fusion Proteins ◽  
Side Population ◽  
Promyelocytic Leukemia ◽  
Molecular Remission ◽  
Nb4 Cells

Abstract Acute promyelocytic leukemia (APL) is distinguished from other AMLs by cytogenetic, clinical, as well as biological characteristics. The hallmark of APL is the t(15;17) which leads to the expression of the PML/RAR fusion protein. PML/RAR is the central leukemia-inducing lesion in APL and is directly targeted by all trans retinoic acid (t-RA). Patients suffering from APL undergo complete hematologic but not molecular remission upon treatment with t-RA. Virtually all patients treated with t-RA-monotherapy had a rapid relapse within few months. But in the combination with an anthracycline, such as doxorubicin or idarubicin, t-RA improved the long term outcome of APL-patients dramatically. Nothing is known about why t-RA-monotherapy is unable to eradicate completely the leukemic population and how it increases the response to chemotherapy. In vitro, the exposure of early hemopoietic stem cells (HSCs) to t-RA does not induce differentiation but selects immature progenitors. Moreover, mice lacking the t-RA-specific receptor RARalpha do not exhibit an impairment of granulopoiesis or hemopoiesis. The indication, that t-RA may be involved in the hemopoietic differentiation, is given by the HL-60 cell line which undergoes granulocytic differentiation at the pharmacological dosages (10−6M) of t-RA. Furthermore vitamin A-deficient mice or mice treated with a antagonist of t-RA accumulate more immature granulocytes in the bone marrow. PML/RAR mediates the response of APL blasts to t-RA, but it is completely unclear, which effect t-RA exerts on the PML/RAR-positive leukemic stem cells which maintains the blast population and represents the source of relapse. Therefore we investigated the effect of t-RA on a cell population with stem cell capacity expressing PML/RAR isolated from the APL cell line NB4 as well as from CD34+/CD38- KG-1 cells transfected with PML/RAR. Here we report that i) the NB4 cells engrafted in NOD/SCID mice indicating the presence of a subpopulation with stem cell capacity in NB4 cells; ii) NB4 had a Hoechst 3342 excluding side population (SP) representing about 1% of the whole cell population; iii) t-RA reduced but did not deplete the side population in NB4 cells; iv) the expression of PML/RAR increased CD34+/CD38- population in KG-1 cells from 75% to over 95%; v) t-RA reduced the CD34+/CD38- population from 75% to 3,5% in mock transfected KG-1 confirming its capacity to induce differentiation, whereas in PML/RAR-positive KG-1 cells it led only to a reduction from 98% to a 25%, which still maintain the capacity to engraft in NOD-SCID mice; vi) also the expression of other fusion proteins, such as AML-1/ETO or PLZF/RAR, associated with t-RA-resistant AML-subtypes, increased the percentage of CD34+/CD38- KG-1 cells over 90%, which was reduced by t-RA only to 35% and 19%, respectively. Taken together these data suggest that a subset of early HSC expressing PML/RAR exhibit the same t-RA-resistant phenotype as HSC expressing fusion proteins associated with AML-subtypes which, in contrast to APL, do not respond to t-RA. These data may give an explanation, why APL-patients do not achieve complete molecular remission upon t-RA monotherapy and undergo early relapse.

In Vivo Analysis of the Role of C/EBPα in Acute Promyelocytic Leukemia Genesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1937-1937
Author(s):  
Barbara A.A. Santana-Lemos ◽  
Florence Guibal ◽  
Maria-Carolina Pintao ◽  
Priscila S. Scheucher ◽  
Rodrigo S. Abreu-Lima ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Leucine Zipper ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Leukemic Cells ◽  
Spleen Cells ◽  
Specific Subset

Abstract Acute promyelocytic leukemia (APL) is characterized by the infiltration of bone marrow (BM) and peripheral blood (PB) by leukemic cells presenting a block of differentiation at the stage of promyelocytes. At the cytogenetic level, APL is associated with the t(15;17) which causes the fusion of two genes: Retinoic Receptor α (RARα) and Promyelocytic Leukemia (PML), on chromosomes 17 and 15, respectively. C/EBPα is a leucine zipper transcription factor essential to normal granulopoiesis. Mutations in C/EBPα gene are detected in 6–10% of Acute Myeloid Leukemia (AML) cases and C/EBPα activity is down-regulated by AML1-ETO fusion protein associated with FAB M2 subtype. We decided to test whether PML-RARα interferes with C/EBPα function, thus contributing to the blockage of differentiation characteristic of APL. We generated mutant mice expressing PML-RARα and haploinsufficient for C/EBPα (PR C/EBPα+/−) by crossing hCG PML-RARα transgenic mice (PR TM) and C/EBPα+/−. Leukemia was not detected in WT (n=415) and C/EBPα+/− (n=47) mice after 800 days of follow up. In contrast, 8.2% PR TM (19/233) and 14.9% (13/87) PR C/EBPα+/− mice developed a form of leukemia that closely resembled human APL and identical to that developed by PR TM. The leukemia-free survival was significantly shorter in PR C/EBPα+/− compared to PR TM (644.3 days; 95% Confidence Interval, 95%C.I.: 586.4 – 702.2 vs 718.4 days; 95%C.I.: 689.3 – 747.5, P=0.02). Both groups presented a long latency for the development of the disease with a mean age (95%C.I.) at diagnosis of 399.9 days (184–673) and 495.8 days (215–757) in PR and PR C/EBPα+/−, respectively. PR and PR C/EBPα+/− leukemic mice presented similar WBC counts (107.1 ± 82.65x103vs 63.55 ± 57.82x103cells/ml, P=0.26), hemoglobin concentrations (10.87 ± 3.69 vs 9.92 ± 2.39 g/dl, P =0.57) and platelet counts (283.8 ± 188.7x103vs 177.8 ± 149.5x103platelets/ml, P =0.24). In both groups, the leukemic cells resembled promyelocytes, and expressed the phenotype CD11b+ Gr1+ CD34± c-Kit+, which represented 46.65 ± 26.89%; 32.72 ± 15.16% and 1.91 ± 1.42% of the spleen cells from PR; PR C/EBPα+/− and WT, respectively. In order to isolate PML-RARα leukemic cells and their normal counterparts to gene expression assessment, BM samples from WT mice (pooled from 2 mice), non leukemic PR C/EBPα+/− mice (pooled from 2 mice), and spleen cells from WT and leukemic PR C/EBPα+/− mice were first submitted to red cells lyses, stained with previously conjugated antibodies. Cells expressing CD16/32, CD11b, c-kit and CD34, but neither CD3 nor CD45/B220 were isolated. These cells presented medium to large size with a granular cytoplasm. C/EBPα and PML-RARα expression was analyzed by qRT-PCR in the sorted cells. Compared to WT cells, promyelocytes from leukemic and non leukemic PR C/EBPα+/− mice expressed significantly less C/EBPα, with the lowest levels detected in leukemic samples. As expected, PML-RARα was not detected in WT samples. Comparison between cells suspensions containing similar numbers of promyelocytes revealed that PML-RARα expression was higher in leukemic compared to non leukemic PR C/EBPα+/− mice. In conclusion, our data strongly suggest that PML-RARα fusion protein acts as a dominant negative product on C/EBPα gene expression in a specific subset of early myeloid cells and contribute to the pathogenesis of APL.

MiRNA Transcription Is Regulated by the PML-RARα Oncoprotein in Acute Promyelocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4197-4197
Author(s):  
Jeannet Nigten ◽  
Ruth Knops ◽  
Gorica Nikoloski ◽  
Theo M. de Witte ◽  
Bert A. van der Reijden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Retinoic Acid ◽  
Cell Differentiation ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Mirna Expression ◽  
Promyelocytic Leukemia ◽  
Mirna Genes ◽  
Before And After

Abstract The discovery of the microRNA (miRNA) molecules has led to new insights into the regulation of gene expression. They are able to bind specific mRNA sequences, and due to inhibition of translation or mRNA degradation, miRNAs cause downregulation of their target genes. To date, several hundreds of unique human miRNAs have been described. So far, for only few of them the target mRNAs have been experimentally confirmed. MiRNAs have been linked to several important biological processes as early stage development, cell growth, cell differentiation and apoptosis. In addition, impaired miRNA expression has been implicated in tumorigenesis. Leukemia is often associated with mutated transcription factors and, as a consequence, deregulated gene expression and impaired proliferation and differentiation. Acute Promyelocytic Leukemia (APL), is characterised by the expression of the mutated transcription factor PML-RARα, which may interfere with the normal function of the retinoic acid receptor α (RARα), a nuclear hormone receptor that acts as a ligand-dependent transcription factor. APL is uniquely sensitive to treatment with the RARα ligand, all-trans retinoic acid (ATRA), which results in the expression of genes that induce terminal granulocytic differentiation of the leukemic blasts. To investigate whether miRNA expression is regulated by ATRA in APL, we performed Taqman miRNA assays for 157 different mature miRNAs in the APL cell line NB4 before and after treatment with ATRA. We found that ATRA induced a more than 10 fold upregulation of 18 miRNAs and a more than 10 fold downregulation of 2 miRNAs. These expression patterns were confirmed in primary APL patient cells before and after treatment with ATRA. To study whether the miRNA expression pattern was dependent on the PML-RARα fusion protein, we used U937 cells stably transfected with a zinc-inducible PML-RARα expression cassette (U937PR9, a kind gift of Dr Pelicci). Upon ATRA treatment, we found that several miRNAs were only induced in the presence of PML-RARα, suggesting that PML-RARα is implicated in the expression of these miRNAs. To investigate whether the PML-RARα fusion protein binds to the endogenous miRNA genes chromatin immunoprecipitation assays were performed with PML-RARα transfected 293 cells. We demonstrated the presence of PML-RARα protein on the miRNA genes. This indicates that the oncoprotein PML-RARα directly influences the expression of these miRNAs. The function of the PML-RARα targeted miRNAs in APL cell differentiation is currently being studied using retroviral expression vectors.

The acute promyelocytic leukemia-specific PML-RARα fusion protein inhibits differentiation and promotes survival of myeloid precursor cells

Cell ◽  
1993 ◽  
Vol 74 (3) ◽  
pp. 423-431 ◽  
Author(s):  
Francesco Grignani ◽  
Pier Francesco Ferrucci ◽  
Ugo Testa ◽  
Giampaolo Talamo ◽  
Marta Fagioli ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Precursor Cells
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