ABSTRACT
A construct for tagging neurospheres and monitoring cell transplantations was developed using a new technology for producing luminescent and radiolabeled probes that have identical structures. The HIV1-Tat basic domain derivatives NAcGRKKRRQRRR(SAACQ)G (SAACQ-1) and [NAcGRKKRRQRRR(Re(CO)3SAACQ)G]+ (ReSAACQ-1) were prepared in excellent yields using the single amino acid chelate-quinoline (SAACQ) ligand and its Re(I) complex and conventional automated peptide synthesis methods. The distribution of the luminescent Re probe, using epifluorescence microscopy, showed that it localized primarily in the cell nucleus with a significant degree of association on the nuclear envelope. A smaller amount was found to be dispersed in the cytoplasm. The 99m Tc analogue was then prepared in 43+/-7% (n=12) yield and very high effective specific activity. Following incubation, average uptake of the probe in neurospheres ranged between 10 and 20 Bq/cell. As determined by colorimetric assays, viability for cells labeled with high effective specific activity 99m TcSAACQ-1 was 97+/-4% at 2 h postlabeling and 85+/-25% at 24 h postlabeling for incubation activities ranging from 245 to 8900 Bq/cell. DNA analysis showed that at these levels, there was no significant difference between the extent of DNA damage in the treated cells versus control cells. A series of preliminary SPECT/CT studies of transplants in mice were performed, which showed that the strategy is convenient and feasible and that it is possible to routinely assess procedures noninvasively and determine the number of cells transplanted.
Subject(s)
Luminescent Proteins/chemical synthesis , Luminescent Proteins/pharmacokinetics , Nervous System , Radiopharmaceuticals/chemical synthesis , Radiopharmaceuticals/pharmacokinetics , Stem Cell Transplantation , Stem Cells/cytology , Animals , Cells, Cultured , Chelating Agents/chemistry , Cytoplasm/diagnostic imaging , Cytoplasm/metabolism , Fluorescent Dyes/chemistry , Head/diagnostic imaging , Head/pathology , Isotope Labeling , Mice , Nervous System/cytology , Nervous System/diagnostic imaging , Nuclear Envelope/diagnostic imaging , Nuclear Envelope/metabolism , Quinolines/chemistry , Staining and Labeling/methods , Stem Cells/diagnostic imaging , Technetium , Tomography, Emission-Computed, Single-Photon , Tomography, X-Ray Computed , Whole Body Imaging , tat Gene Products, Human Immunodeficiency Virus/chemistry , tat Gene Products, Human Immunodeficiency Virus/pharmacokineticsABSTRACT
The number of the nucleoli in a CaCo-2 cell nucleus does not generally depend on the quantity of DNA in the nucleus, but nucleolar DNA content is directly proportional to total nuclear DNA. However, in multinucleolar cells (three or more nucleoli), the nucleolar DNA content increases after 96 h incubation in culture without concomitant quantitative changes in nuclear DNA. The percentage of multinucleolar cells and the average number of nucleoli per nucleus increase with increasing incubation time. After 72 and 96 h in culture, multinucleolar cells show distinctive morphologies. The ratio of the sum of nucleolar perimeters to the nuclear perimeter increases linearly when the number of nucleoli in a nucleus increases, but there is no concomitant increase in total nucleolar area or DNA content, except in the 72 and 96 h populations. When the number of nucleoli in CaCo-2 cells increases after 48 and 60 h in culture, the amount of DNA per nucleolus decreases.
