To facilitate molecular genetic analysis of vertebrate development, haploid genetics was used to construct a recombination map for the zebrafish Danio (Brachydanio) rerio. The map consists of 401 random amplified polymorphic DNAs (RAPDs) and 13 simple sequence repeats spaced at an average interval of 5.8 centimorgans. Strategies that exploit the advantages of haploid genetics and RAPD markers were developed that quickly mapped lethal and visible mutations and that placed cloned genes on the map. This map is useful for the position-based cloning of mutant genes, the characterization of chromosome rearrangements, and the investigation of evolution in vertebrate genomes.

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a variety of tumor types and has been targeted with radiolabeled peptides for detection and therapy of these cancers. Analogues of the 14 amino acid bombesin (BN) peptide have been radiolabeled with both gamma- and positron-emitting radionuclides for detection of GRPR-expressing tumors. We have previously evaluated BN analogues radiolabeled with the positron-emitter, copper-64 (64Cu), that contained various aliphatic linkers placed between the BN peptide and the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. These studies showed that the analogues could be used for positron-emission tomographic (PET) imaging of GRPR-positive tumors in mice but clinical translation would be hindered by significant uptake in background tissues. Therefore, the purpose of this study was to determine if the use of amino acid linkers placed between the DOTA chelate and the BN peptide would reduce nontarget tissue uptake, while maintaining good prostate tumor uptake. The linkers studied utilized three amino acid combinations of glycine (G), serine (S), or glutamic acid (E). In vitro assays in PC-3 cells showed that the glutamic acid-containing linkers had poor binding and internalization, while the other analogues had IC50 values <100 nM and good internalization. In vivo, these same analogues demonstrated tumor-specific uptake and good imaging characteristics that were comparable to, or better than the previously reported 64Cu-labeled DOTA-BN analogues. Overall, this study shows that BN analogues containing amino acid linkers can be used for the PET imaging of GRPR-expressing prostate cancer and that these linkers lead to lower background tissue uptake.

Mammography is a well-established method for detecting primary breast cancer; however, it has some limitations that may be overcome using nuclear imaging methods. Current radiopharmaceuticals have limited sensitivity for detecting small primary lesions and it has been suggested that novel radiopharmaceuticals are necessary for detection of primary breast cancer, as well as for detecting metastases and recurrence, or for monitoring therapy. The gastrin-releasing peptide receptor (GRPR) is a seven-transmembrane G-protein coupled receptor that is overexpressed on primary breast cancer and lymph node metastases. Bombesin (BN) is a tetradecapeptide that binds with high affinity to GRPR and can be radiolabeled with the positron-emitter, copper-64 ((64)Cu) for imaging with positron-emission tomography (PET). The goal of this study was to evaluate BN analogs that could be radiolabeled with (64)Cu for PET imaging of breast cancer. A series of BN analogs containing 4, 5, 6, 8, and 12- carbon linkers were evaluated with regard to their binding and internalization into T-47D human breast cancer cells. The (64)Cu-labeled analogs were then evaluated in mice bearing T-47D xenografts by tissue biodistribution and microPET imaging. These studies showed that all of the analogs had IC(50) values <100 nM and were all internalized into T-47D cells. Biodistribution studies showed that the BN analog with the 8-carbon linker not only had the highest tumor uptake but also had high normal tissue uptake in the liver. The analogs containing the 6- or 8-carbon linkers demonstrated good tumor uptake as determined by microPET imaging. Overall, this study shows the feasibility of using positron-labeled BN analogs for PET detection of GRPR-expressing breast cancer.

Bombesin is a 14–amino-acid amphibian peptide that binds with high affinity to the gastrin-releasing peptide receptor (GRPR), which is overexpressed on a variety of solid tumors. It has been demonstrated that bombesin analogs can be radiolabeled with a variety of radiometals for potential diagnosis and treatment of GRPR-positive tumors. In this regard, several studies have used different chelators conjugated to the 8 C-terminal amino acids of bombesin(7–14) for radiolabeling with 64Cu. These analogs have demonstrated GRPR-specific small-animal PET of tumors but have various advantages and disadvantages. The objective of this study was to conjugate the previously described (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) (SarAr) chelator to bombesin (7–14), radiolabel the conjugate with 64Cu, and evaluate in vitro and in vivo. Methods SarAr was synthesized as previously published and conjugated to bombesin(7–14) by solid-phase peptide synthesis using standard Fmoc chemistry. Succinic acid (SA), 8-aminooctanoic acid (Aoc), and Gly-Ser-Gly (GSG) were used as linkers between SarAr and bombesin(7–14) to yield the resulting SarAr-SA-Aoc-bombesin(7–14) and SarAr-SA-Aoc-GSG-bombesin(7–14) peptides. The unlabeled peptides were evaluated in a competitive binding assay using PC-3 prostate cancer cells and 125I-Tyr4-bombesin to determine the inhibitory concentration of 50%. The peptides were radiolabeled with 64Cu and evaluated for internalization into PC-3 cells in vitro and for in vivo tumor uptake in mice bearing PC-3 xenografts using biodistribution and small-animal PET/CT studies. Results The competitive binding assay demonstrated that both SarAr-SA-Aoc-bombesin(7–14) and SarAr-SA-Aoc-GSG-bombesin(7–14) bound with high affinity to GRPR with an inhibitory concentration of 50% of 3.5 and 4.5 nM, respectively. Both peptides were radiolabeled with 64Cu at room temperature without further purification and demonstrated similar internalization into PC-3 cells. In vivo, the radiolabeled peptides demonstrated tumor-specific uptake (13.0 and 8.5 percentage injected dose per gram for 64Cu-SarAr-SA-Aoc-bombesin(7–14) and 64Cu-SarAr-SA-Aoc-GSG-bombesin(7–14), respectively, at 1 h) and imaging that was comparable to, or better than, that of the previously reported 64Cu-labeled bombesin analogs. The 64Cu-SarAr-SA-Aoc-GSG-bombesin(7–14) had more rapid blood clearance and lower tumor and normal-tissue uptake than 64Cu-SarAr-SA-Aoc-bombesin(7–14), resulting in similar tumor-to-blood ratios for each analog (15.1 vs. 11.3 for 64Cu-SarAr-SA-Aoc-bombesin(7–14) and 64Cu-SarAr-SA-Aoc-GSG-bombesin(7–14), respectively, at 1 h). Conclusion These studies demonstrate that 64Cu-SarAr-SA-Aoc-bombesin(7–14) and 64Cu-SarAr-SA-Aoc-GSG-bombesin(7–14) bound with high affinity to GRPR-expressing cells and that these peptides can be used for PET of GRPR-expressing prostate cancer.

Highlights d NS1 partitions into the nucleus upon RSV infection d NS1 coimmunoprecipitates with chromatin-associated proteins d NS1 binds regulatory elements of differentially expressed genes in RSV infection d NS1 modulates host gene transcription