title: Novel cell nucleus directed fluorescent tetraazacyclododecane-tetra-acetic acid compounds.
abstract: Peptide conjugates derived from the SV 40 T antigen nuclear localisation sequence (NLS) have been successfully used to translocate both fluorescein isothiocyanate (FITC) and Gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) into the cytoplasm and nucleus of glioma cells. However, uptake occurred only in up to 35% of cells. To improve cellular uptake, we designed three novel FITC-labelled Gd-DOTA conjugates. In the first conjugate, the commonly used Gd-DOTA-complex was coupled to the nuclear localization sequence (NLS) of the Simian Virus (SV) 40 T antigen alone as a control. In the second conjugate, the Gd-DOTA-coupled SV 40 T antigen NLS was elongated by the HIV-1 tat peptide (HIV-NLS). A third conjugate, in which the Gd-DOTA-complex was coupled to the SV 40 T antigen NLS elongated by a peptide containing seven arginines and six aminohexanoic acids (Ahx6R7) was also synthesized (AHX-NLS). By means of confocal laser scanning microscopy, fluorescence activated cell sorting, magnetic resonance imaging (MRI) and viability tests we were able to demonstrate that the first conjugate containing only the NLS of the SV 40 T antigen stained the nuclei of no more than 10-12% of U373 and LN18 glioma cells, resulting in low signal intensity in MRI. The stained cells remained viable. After incubation with conjugates HIV-NLS and AHX-NLS the nuclei of up to 73% of U373 and LN18 glioma cells were stained. This was associated with high signal intensity in MRI and cell death. As previously shown, the gadolinium ion reduces cellular uptake of DOTA conjugates. To confirm this, the conjugates were produced with or without gadolinium. The gadolinium-free DOTA conjugates showed a higher cellular uptake rate and an increased cytotoxic potential.
authors.affiliation: Department of Neuroradiology, University of Tübingen, Hoppe-Seyler-Str.3, 72076 Tübingen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Sturzu
authors.forename: U
authors.initials: U
authors.lastname: Klose
authors.forename: H
authors.initials: H
authors.lastname: Echner
authors.forename: M
authors.initials: M
authors.lastname: Regenbogen
authors.forename: H
authors.initials: H
authors.lastname: Kalbacher
authors.forename: A
authors.initials: A
authors.lastname: Gharabaghi
authors.forename: S
authors.initials: S
authors.lastname: Heckl
mesh_terms: D021581:Active Transport, Cell Nucleus; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D003593:Cytoplasm / Q000378:metabolism; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D005910:Glioma / Q000473:pathology; D006571:Heterocyclic Compounds / Q000378:metabolism / Q000494:pharmacology; D006573:Heterocyclic Compounds, 1-Ring / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008279:Magnetic Resonance Imaging; D018613:Microscopy, Confocal; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism
chemical_list: D000952:Antigens, Polyomavirus Transforming; D005456:Fluorescent Dyes; D006571:Heterocyclic Compounds; D006573:Heterocyclic Compounds, 1-Ring; D019913:Nuclear Localization Signals; D009942:Organometallic Compounds; D010455:Peptides; D054322:tat Gene Products, Human Immunodeficiency Virus; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; C050823:gadolinium 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetate; D016650:Fluorescein-5-isothiocyanate