title: Protein Kinase C-α is a Critical Protein for Antisense Oligonucleotide-mediated Silencing in Mammalian Cells.
abstract: We have identified the existence of a productive, PKC-α-dependent endocytotic silencing pathway that leads gymnotically-delivered locked nucleic acid (LNA)-gapmer phosphorothioate antisense oligonucleotides (ASOs) into late endosomes. By blocking the maturation of early endosomes to late endosomes, silencing the expression of PKC-α results in the potent reduction of ASO silencing ability in the cell. We have also demonstrated that silencing of gene expression in the cytoplasm is vitiated when PKC-α expression is reduced. Restoring PKC-α expression via a reconstitution experiment reinstates the ability of ASOs to silence. These results advance our understanding of intracellular ASO trafficking and activity following gymnotic delivery, and further demonstrate the existence of two distinct silencing pathways in mammalian cells, one in the cytoplasmic and the other in the nuclear compartment.
authors.affiliation: Department of Medical Oncology, City of Hope, Duarte, California, USA. Electronic address: dcastanotto@coh.org.
authors.forename: Daniela
authors.initials: D
authors.lastname: Castanotto
authors.affiliation: Department of Cancer Immunotherapeutics and Tumor Immunology, City of Hope, Duarte, California, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Lin
authors.affiliation: Department of Molecular Medicine, City of Hope, Duarte, California, USA.
authors.forename: Claudia
authors.initials: C
authors.lastname: Kowolik
authors.affiliation: Roche Innovation Center Copenhagen, Horsholm, Denmark.
authors.forename: Troels
authors.initials: T
authors.lastname: Koch
authors.affiliation: Roche Innovation Center Copenhagen, Horsholm, Denmark.
authors.forename: Bo Rode
authors.initials: BR
authors.lastname: Hansen
authors.affiliation: Roche Innovation Center Copenhagen, Horsholm, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Oerum
authors.affiliation: Department of Medical Oncology, City of Hope, Duarte, California, USA. Electronic address: cstein@coh.org.
authors.forename: Cy A
authors.initials: CA
authors.lastname: Stein
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D011992:Endosomes / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D009841:Oligonucleotides / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors* / Q000235:genetics; D013873:Thionucleotides
chemical_list: D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D013873:Thionucleotides; C477371:locked nucleic acid; C496628:PRKCA protein, human; D051571:Protein Kinase C-alpha