title: Value of apoptin's 40-amino-acid C-terminal fragment for the differentiation between human tumor and non-tumor cells.
abstract: Apoptin, a protein of the chicken anemia virus (CAV), consists of 121 amino acids (aa) and represents a novel, potentially tumor-specific therapeutic and diagnostic agent. The C-terminal part of Apoptin (aa 81-121) is believed to contain a bipartite nuclear localization signal (NLS) (NLS1: aa 82-88 and NLS2: aa 111-121), which is only active in tumor cells after phosphorylation of threonine(108) by tumor-specific cytoplasmic phosphokinases. Furthermore, a nuclear export signal (NES) (aa 97-105) seems to enable nuclear export of Apoptin only in healthy cells. The specificity for tumor cell nuclei also applies to the truncated C-terminal part of Apoptin (aa 81-121), which therefore represents a highly attractive peptide sequence for peptide synthesis. Here we describe for the first time the synthesis of fluorescein isothiocyanate (FITC)- and Dansyl-labelled conjugates containing this C-terminal part of Apoptin, with either phosphorylated or nonphosphorylated threonine(108). The phosphorylated conjugates were synthesized in an attempt to achieve nuclear accumulation in healthy cells, which lack cytoplasmic tumor-specific phosphokinases. Surprisingly, all the conjugates accumulated rapidly within the cell nuclei of both tumor and non-tumor cells from the bladder, brain and prostate and led to cell death. By coupling Apoptin(81-121) to FITC and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) at either the C- or N-terminus we could exlude that the coupling site is decisive for tumor cell-specific nuclear localization. The labels FITC, DOTA and Dansyl were not responsible for cell death in healthy cells because cell death was not prevented by using an unlabelled Apoptin(81-121) peptide. Cellular and nuclear uptake of the FITC-labelled Apoptin(81-121) peptide was almost completely abolished after altering the NLS2 (replacement of five arginines with serines).
authors.affiliation: Department of Neuroradiololgy, University of Tübingen, Medical School, Hoppe-Seyler-Str. 3, 72076, Tübingen, Germany. stefan.heckl@med.uni-tuebingen.de
authors.forename: Stefan
authors.initials: S
authors.lastname: Heckl
authors.forename: Marc
authors.initials: M
authors.lastname: Regenbogen
authors.forename: Alexander
authors.initials: A
authors.lastname: Sturzu
authors.forename: Alireza
authors.initials: A
authors.lastname: Gharabaghi
authors.forename: Gerhard
authors.initials: G
authors.lastname: Feil
authors.forename: Alexander
authors.initials: A
authors.lastname: Beck
authors.forename: Hartmut
authors.initials: H
authors.lastname: Echner
authors.forename: Thomas
authors.initials: T
authors.lastname: Nagele
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects*; D001253:Astrocytes / Q000648:ultrastructure; D001921:Brain / Q000648:ultrastructure; D036022:Capsid Proteins / Q000737:chemistry; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003619:Dansyl Compounds / Q000737:chemistry; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005910:Glioma / Q000175:diagnosis; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D006801:Humans; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D049790:Nuclear Export Signals / Q000502:physiology; D019913:Nuclear Localization Signals / Q000502:physiology; D010446:Peptide Fragments; D010769:Phosphothreonine / Q000737:chemistry / Q000378:metabolism; D011467:Prostate / Q000648:ultrastructure; D011471:Prostatic Neoplasms / Q000175:diagnosis; D001743:Urinary Bladder / Q000648:ultrastructure; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis; D019459:Urothelium / Q000648:ultrastructure
chemical_list: D036022:Capsid Proteins; D003619:Dansyl Compounds; D006573:Heterocyclic Compounds, 1-Ring; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D010446:Peptide Fragments; C091717:VP3 protein, Chicken anemia virus; D010769:Phosphothreonine; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D016650:Fluorescein-5-isothiocyanate