Localization of iodine-125-mIP-Des-Met14-bombesin (7-13)NH2 in ovarian carcinoma induced to express the gastrin releasing peptide receptor by adenoviral vector-mediated gene transfer.

UNLABELLED: The gastrin releasing peptide receptor (GRPr) has a high affinity for the 14 amino acid bombesin peptide. For this analysis, [125I]-Tyr4-bombesin was compared with [125I]-mIP-bombesin (a seven amino acid bombesin analog) for in vitro binding and internalization into tumor cells and for tumor localization in vivo. Also, a recombinant adenoviral vector (AdCMVGRPr) was used for gene transfer to induce the expression of GRPr in human ovarian cancer cells for binding and tumor localization with these radiolabeled peptides.
METHODS: [125I]-mIP-bombesin was synthesized and compared with [125I]-Tyr4-bombesin in internalization assays using BNR-11 cells (mouse fibroblast cells stably transfected with GRPr) over a 24-hr period. In vitro binding assays used BNR-11, and A427, HeLa and SKOV3.ip1 human cancer cells, which were either uninfected or infected with AdCMVGRPr. Biodistribution studies were performed in normal BALB/c mice and in athymic nude mice bearing orthotopic SKOV3.ip1 ovarian cancer tumors. The SKOV3.ip1 tumors were induced to express GRPr with the AdCMVGRPr adenoviral vector.
RESULTS: Internalization assays showed that [125I]-Tyr4-bombesin was rapidly internalized and catabolized at 37 degrees C with approximately 10% of the radioactivity remaining intracellularly at 4 hr, compared with approximately 30% with [125I]-mIP-bombesin. HeLa, A427 and SKOV3.ip1 cells were all induced to express levels of GRPr that were higher than those seen with the positive control BNR-11 cells. Normal mice showed a lower level of radioactivity in both the blood and thyroid for [125I]-mIP-bombesin [0.26% +/- 0.10% injected dose per gram (ID/g) and 0.24% +/- 0.05% ID] than for [125I]-Tyr4-bombesin (3.5% +/- 1.6% ID/g and 5.2% +/- 4.4% ID) at 4 hr postinjection. Mice bearing intraperitoneal (i.p.) SKOV3.ip1 tumors and given AdCMVGRPr i.p. 5 days after tumor cell inoculation followed by [125I]-mIP-bombesin i.p. at day 7 showed 16.5% +/- 4.8% ID/g in tumor compared with 5.9% +/- 3.0% ID/g with [125I]-Tyr4-bombesin at 4 hr postinjection. Tumor bearing mice given saline or a control adenovirus expressing the beta-galactosidase (LacZ) gene showed significantly lower tumor uptake values of both bombesin peptides.
CONCLUSION: Internalization assays showed that [125I]-mIP-bombesin has favorable characteristics compared with [125I]-Tyr4-bombesin with regards to cellular internalization and retention. The results demonstrate successful in vitro and in vivo transduction of human tumor cells with a recombinant adenoviral vector-expressing GRPr. Additionally, tumors transduced in vivo to express GRPr demonstrated significantly greater localization of [125I]-mIP-bombesin when compared with [125I]-Tyr4-bombesin.