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1.
Hum Gene Ther ; 22(12): 1593-8, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21846246

ABSTRACT

Acute promyelocytic leukemia (APL) results from a chromosomal translocation that gives rise to the leukemogenic fusion protein PML-RARα (promyelocytic leukemia-retinoic acid α receptor). Differentiation of leukemic cells and complete remission of APL are achieved by treatment of patients with pharmacological doses of all-trans retinoic acid (ATRA), making APL a model disease for differentiation therapy. However, because patients are resistant to further treatment with ATRA on relapse, it is necessary to develop alternative treatment strategies to specifically target APL. We therefore sought to develop a treatment strategy based on lentiviral vector-mediated delivery of small interfering RNA (siRNA) that specifically targets the breakpoint region of PML-RARα. Unlike treatment with ATRA, which resulted in differentiation of leukemic NB4 cells, delivery of siRNA targeting PML-RARα into NB4 cells resulted in both differentiation and apoptosis, consistent with the specific knockdown of PML-RARα. Intraperitoneal injection of NB4 cells transduced with lentiviral vectors delivering PML-RARα-specific siRNA but not control siRNA prevented development of disease in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Taken together, these results indicate that development of PML-RARα-specific siRNA may represent a promising treatment strategy for ATRA-resistant APL.


Subject(s)
Apoptosis , Cell Differentiation , Genetic Vectors/therapeutic use , Lentivirus/genetics , Leukemia, Promyelocytic, Acute/pathology , Leukemia, Promyelocytic, Acute/therapy , Oncogene Proteins, Fusion/antagonists & inhibitors , RNA, Small Interfering/genetics , Animals , Antineoplastic Agents/pharmacology , Blotting, Western , Cells, Cultured , Drug Resistance, Neoplasm , Female , Flow Cytometry , Humans , Injections, Intraperitoneal , Leukemia, Promyelocytic, Acute/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Oncogene Proteins, Fusion/genetics , Tretinoin/pharmacology
2.
Proc Natl Acad Sci U S A ; 108(1): 331-6, 2011 Jan 04.
Article in English | MEDLINE | ID: mdl-21173229

ABSTRACT

Measles virus (MV), a member of the family Paramyxoviridae and an exclusively human pathogen, is among the most infectious viruses. A progressive fatal neurodegenerative complication, subacute sclerosing panencephalitis (SSPE), occurs during persistent MV infection of the CNS and is associated with biased hypermutations of the viral genome. The observed hypermutations of A-to-G are consistent with conversions catalyzed by the adenosine deaminase acting on RNA (ADAR1). To evaluate the role of ADAR1 in MV infection, we selectively disrupted expression of the IFN-inducible p150 ADAR1 isoform and found it caused embryonic lethality at embryo day (E) 11-E12. We therefore generated p150-deficient and WT mouse embryo fibroblast (MEF) cells stably expressing the MV receptor signaling lymphocyte activation molecule (SLAM or CD150). The p150(-/-) but not WT MEF cells displayed extensive syncytium formation and cytopathic effect (CPE) following infection with MV, consistent with an anti-MV role of the p150 isoform of ADAR1. MV titers were 3 to 4 log higher in p150(-/-) cells compared with WT cells at 21 h postinfection, and restoration of ADAR1 in p150(-/-) cells prevented MV cytopathology. In contrast to infection with MV, p150 disruption had no effect on vesicular stomatitis virus, reovirus, or lymphocytic choriomeningitis virus replication but protected against CPE resulting from infection with Newcastle disease virus, Sendai virus, canine distemper virus, and influenza A virus. Thus, ADAR1 is a restriction factor in the replication of paramyxoviruses and orthomyxoviruses.


Subject(s)
Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Embryonic Development/genetics , Mutation/genetics , SSPE Virus/genetics , Subacute Sclerosing Panencephalitis/genetics , Virus Replication/genetics , Animals , Antigens, CD/metabolism , Cell Line , DNA Primers/genetics , Fluorescent Antibody Technique , Gene Knockout Techniques , Green Fluorescent Proteins , Mice , Mice, Inbred C57BL , Protein Isoforms/genetics , RNA-Binding Proteins , Receptors, Cell Surface/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signaling Lymphocytic Activation Molecule Family Member 1
3.
J Biol Chem ; 281(6): 3244-53, 2006 Feb 10.
Article in English | MEDLINE | ID: mdl-16339759

ABSTRACT

The protein kinase regulated by RNA (PKR) is interferon (IFN)-inducible and plays important roles in many cellular processes, including virus multiplication, cell growth, and apoptosis. The TATA-less PKR promoter possesses a novel 15-bp DNA element (kinase conserved sequence (KCS)) unique to the human and mouse PKR genes that is conserved in sequence and position. We found that Sp1 and Sp3 of the Sp family of transcription factors bind at the KCS element. Their involvement was analyzed in the activation of basal and IFN-inducible PKR promoter activity. Both the small and large isoforms of Sp3 co-purified with KCS protein binding activity (KBP) by using nuclear extracts from HeLa cells not treated with IFN. Two forms of the KCS-binding protein complex were demonstrated by electrophoretic mobility shift assay analysis; one contained Sp1 and the other Sp3. In mouse cells null for all Sp3 isoforms, PKR expression was reduced to approximately 50% that of wild-type cells in the absence of IFN. The IFN-inducible expression of PKR, however, was Sp3-independent but STAT1- and JAK1-dependent. Overexpression of Sp1 in human U cells resulted in increased PKR promoter activity. In Drosophila SL2 cells lacking Sp proteins, both Sp1 and Sp3 large but not small isoforms activated PKR promoter expression, with the Sp1-mediated activation dominant. Mutational analysis of the PKR promoter region indicated a cooperative interaction between two different Sp sites, one of which is within the KCS element. These results establish that, in the absence of IFN treatment, activation of PKR basal expression is mediated by Sp1 and Sp3 proteins in a cooperative manner.


Subject(s)
Interferons/metabolism , Promoter Regions, Genetic , Sp Transcription Factors/metabolism , Sp1 Transcription Factor/metabolism , Sp3 Transcription Factor/metabolism , eIF-2 Kinase/genetics , Animals , Apoptosis , Base Sequence , Blotting, Western , Cell Line , Cell Nucleus/metabolism , DNA Mutational Analysis , Drosophila , Drosophila melanogaster , Fibroblasts/metabolism , HeLa Cells , Humans , Mice , Models, Biological , Models, Genetic , Molecular Sequence Data , Plasmids/metabolism , Protein Binding , Protein Isoforms , Transcription Factors , Transcriptional Activation , Transfection
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