ABSTRACT
Acute promyelocytic leukemia (APL) is characterized by a block in differentiation and accumulation of promyelocytes in the bone marrow and blood. The majority of APL patients harbor the t(15:17) translocation leading to expression of the fusion protein promyelocytic-retinoic acid receptor alpha. Treatment with retinoic acid leads to degradation of promyelocytic-retinoic acid receptor alpha protein and disappearance of leukemic cells; however, 30% of APL patients relapse after treatment. One potential mechanism for relapse is the persistence of cancer "stem" cells in hematopoietic organs after treatment. Using a novel sorting strategy we developed to isolate murine myeloid cells at distinct stages of differentiation, we identified a population of committed myeloid cells (CD34(+), c-kit(+), FcgammaRIII/II(+), Gr1(int)) that accumulates in the spleen and bone marrow in a murine model of APL. We observed that these cells are capable of efficiently generating leukemia in recipient mice, demonstrating that this population represents the APL cancer-initiating cell. These cells down-regulate the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) possibly through a methylation-dependent mechanism, indicating that C/EBPalpha deregulation contributes to transformation of APL cancer-initiating cells. Our findings provide further understanding of the biology of APL by demonstrating that a committed transformed progenitor can initiate and propagate the disease.
Subject(s)
Bone Marrow Cells/pathology , Leukemia, Promyelocytic, Acute/pathology , Myeloid Cells/pathology , Neoplastic Stem Cells/pathology , Animals , Antigens, CD34/metabolism , Bone Marrow Cells/metabolism , CCAAT-Enhancer-Binding Protein-alpha/genetics , Calgranulin A/genetics , Calgranulin A/metabolism , Cell Line, Tumor , DNA Methylation , Female , Flow Cytometry , Humans , Kaplan-Meier Estimate , Leukemia, Promyelocytic, Acute/blood , Leukemia, Promyelocytic, Acute/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Cells/metabolism , Neoplastic Stem Cells/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-kit/metabolism , U937 CellsABSTRACT
The transcription factor PU.1 is an important regulator of hematopoiesis; precise expression levels are critical for normal hematopoietic development and suppression of leukemia. We show here that noncoding antisense RNAs are important modulators of proper dosages of PU.1. Antisense and sense RNAs are regulated by shared evolutionarily conserved cis-regulatory elements, and we can show that antisense RNAs inhibit PU.1 expression by modulating mRNA translation. We propose that such antisense RNAs will likely be important in the regulation of many genes and may be the reason for the large number of overlapping complementary transcripts with so far unknown function.
Subject(s)
Gene Expression , Proto-Oncogene Proteins/genetics , RNA, Antisense/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Trans-Activators/genetics , Animals , Cell Line , Electroporation , Granulocytes/cytology , Granulocytes/metabolism , HL-60 Cells , Humans , Immunomagnetic Separation , Jurkat Cells , Macrophages/metabolism , Mice , Models, Genetic , Protein Biosynthesis , RNA Interference , RNA, Antisense/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , T-Lymphocytes/metabolism , Transcription, Genetic , U937 CellsABSTRACT
The ankyrin repeat-containing protein with a suppressor of cytokine signaling box-2 (ASB2) gene was identified as a retinoic acid-response gene and a target of the promyelocytic leukemia-retinoic acid receptor-alpha oncogenic protein characteristic of acute promyelocytic leukemia. Expression of ASB2 in myeloid leukemia cells inhibits growth and promotes commitment, recapitulating an early step known to be critical for differentiation. Here we show that ASB2, by interacting with the Elongin BC complex, can assemble with Cullin5.Rbx1 to form an E3 ubiquitin ligase complex that stimulates polyubiquitination by the E2 ubiquitin-conjugating enzyme Ubc5. This is a first indication that a member of the ASB protein family, ASB2, is a subunit of an ECS (Elongin C-Cullin-SOCS box)-type E3 ubiquitin ligase complex. Altogether, our results strongly suggest that ASB2 targets specific proteins to destruction by the proteasome in leukemia cells that have been induced to differentiate.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carrier Proteins/metabolism , Cullin Proteins/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line , Elongin , Humans , Leukemia/enzymology , Leukemia/metabolism , Leukemia/pathology , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Mutation , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Processing, Post-Translational , Transcription Factors/genetics , Two-Hybrid System TechniquesABSTRACT
Retinoic acid induces clinical remission in acute promyelocytic leukemia (APL) by triggering differentiation of leukemia promyelocytes. Here, we have characterized a gene encoding a member of the immunoglobulin superfamily, among novel retinoic acid-induced genes identified in APL cells. This protein, which was named JAML (junctional adhesion molecule-like), contains 2 extracellular immunoglobulin-like domains, a transmembrane segment, and a cytoplasmic tail. JAML mRNA is expressed in hematopoietic tissues and is prominently expressed in granulocytes. The fact that JAML protein is localized at the cell plasma membrane in the areas of cell-cell contacts, whereas it is not detected at free cell borders, suggests that JAML is engaged in homophilic interactions. Furthermore, a conserved dimerization motif among JAM members was shown to be important for JAML localization at the cell membrane. Finally, exogenous expression of JAML in myeloid leukemia cells resulted in enhanced cell adhesion to endothelial cells. Altogether, our results point to JAML as a novel member of the JAM family expressed on leukocytes with a possible role in leukocyte transmigration.
Subject(s)
Cell Adhesion Molecules/biosynthesis , Gene Expression Regulation/drug effects , Leukemia, Myeloid/pathology , Base Sequence , Cell Adhesion/drug effects , Cell Adhesion Molecules/genetics , Cell Differentiation/genetics , Cell Line, Tumor , Endothelium, Vascular/cytology , Humans , Junctional Adhesion Molecules , Leukemia, Myeloid/metabolism , Leukocytes/cytology , Leukocytes/metabolism , Molecular Sequence Data , RNA, Messenger/biosynthesis , Sequence Analysis , Tretinoin/pharmacology , Tumor Cells, CulturedABSTRACT
In acute promyelocytic leukemia (APL) cells harboring the promyelocytic leukemia retinoic acid receptor alpha (PML-RARalpha) chimeric protein, retinoic acid (RA)-induced differentiation is triggered through a PML-RARalpha signaling resulting in activation of critical target genes. Induced differentiation of APL cells is always preceded by withdrawal from the cell cycle and commitment events leading to terminal differentiation. Here we have identified the human ankyrin repeat-containing protein with a suppressor of cytokine signaling box-2 (ASB-2) cDNA, as a novel RA-induced gene in APL cells. PML-RARalpha strongly enhanced RA-induced ASB-2 mRNA expression. In myeloid leukemia cells, ASB-2 expression induced growth inhibition and chromatin condensation recapitulating early events critical to RA-induced differentiation of APL cells.
