Characterization and Development of a Peptide (p160) with Affinity for Neuroblastoma Cells
Vasileios Askoxylakis1,2,
Walter Mier2,
Sabine Zitzmann1,
Volker Ehemann3,
Jianbing Zhang4,
Susanne Krämer2,
Carmen Beck2,
Manfred Schwab5,
Michael Eisenhut6 and
Uwe Haberkorn1,2
1 Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center (DKFZ) and University of Heidelberg, Heidelberg, Germany; 2 Department of Nuclear Medicine, University of Heidelberg, Heidelberg, Germany; 3 Institute for Pathology, University of Heidelberg, Heidelberg, Germany; 4 Institute for Biological Sciences, National Research Council, Ottawa, Canada; 5 Department of Tumour Genetics, German Cancer Research Center (DKFZ), Heidelberg, Germany; and 6 Department of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ), Heidelberg, Germany
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FIGURE 1. Binding and competition of 125I-labeled p160. (A) Specific binding of 125I-p160 to neuroblastoma WAC 2 cells. Nonspecific binding was determined in presence of unlabeled p160 (10–4 mol/L). The peptides octreotide and D-p160 were used at same concentration (10–4 mol/L) as negative control competitors. (B) Binding of 125I-p160 with and without excess of unlabeled peptide (10–4 mol/L) in WAC 2 and HUVEC cells. Incubation was performed for 1 h at 37°C. (C) Competition for bound 125I-p160 by unlabeled p160 peptide. WAC 2 cells were incubated with 1–2 x 106 cpm radioligand and increasing concentrations of unlabeled p160 peptide. (D) In vitro cell accumulation of 125I-p160 in WAC 2 cells as function of time. Incubation was performed for periods from 5 to 180 min. All experiments were performed in triplicate; SD is shown.
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FIGURE 2. Fluorescence-activated cell-sorting analysis. (A) Autofluorescence of WAC 2 cells. (B) Fluorescence of WAC 2 cells after incubation with FITC (10–6 mol/L) for 20 min at 37°C. (C) Fluorescence of WAC 2 cells after incubation with FITC-Lys-p160 (10–6 mol/L) for 20 min at 37°C. (D) Fluorescence of WAC 2 cells after incubation with FITC-Lys-p160 (10–6 mol/L) for 20 min in presence of unlabeled p160 at concentration of 10–4 mol/L.
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FIGURE 3. Binding and internalization of 125I-p160 in WAC 2 cells. Cells were grown for 24 h and incubated with 1–2 x 106 cpm radioligand for 1 h at 37°C or at 4°C. After being washed with acid buffer (pH 2.8), cells were lysed and internalized radioactivity was measured.
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FIGURE 4. Binding and competition of 125I-labeled p160 and 125I-ß-Ala-p160-8-2. (A) Specific binding of 125I-p160 and 125I-ß-Ala-p160-8-2 to WAC 2 cells. As competitors, unlabeled p160 and ß-Ala-p160-8-2 were used at concentration of 5 x 10–5 mol/L. Incubation was performed for 1 h at 37°C. (B) Time kinetics of 125I-ß-Ala-p160-8-2 binding to WAC 2. (C) Unlabeled ß-Ala-p160-8-2 was incubated in human serum, and aliquots were removed for subsequent analysis at indicated time points. Experiments A and B were performed in triplicate; SD is shown.
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FIGURE 5. In female BALB/c nu/nu mice carrying WAC 2 tumors, organ distribution of p160 after 1-h circulation of 131I-labeled p160 (n = 4 animals per experiment) and after perfusion (n = 6 animals per experiment).
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FIGURE 6. Time kinetic organ distribution of ßAla-p160-8-2 in female BALB/c nu/nu mice carrying WAC 2 tumors. Incubation was performed for 5, 15, and 60 min (n = 3 animals per experiment) with 131I-labeled ßAla-p160-8-2.
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Copyright © 2006 by the Society of Nuclear Medicine.
