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.
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.


CONCLUSIONS
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.
Rogers
B E
BE
Department of Radiation Oncology, University of Alabama at Birmingham 35294, USA.
Rosenfeld
M E
ME
Khazaeli
M B
MB
Mikheeva
G
G
Stackhouse
M A
MA
Liu
T
T
Curiel
D T
DT
Buchsbaum
D J
DJ
D000256:Adenoviridae; D000818:Animals; D001839:Bombesin / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007457:Iodine Radioisotopes; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010051:Ovarian Neoplasms / Q000000981:diagnostic imaging* / Q000378:metabolism; D010446:Peptide Fragments / Q000493:pharmacokinetics; D011877:Radionuclide Imaging; D018004:Receptors, Bombesin / Q000096:biosynthesis*; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D018004:Receptors, Bombesin; C107634:bombesin(8-13)-NH2, N-(3-iodobenzoyl)glutamyl-des-Met(14)-; C035891:bombesin, Tyr(4)-; D001839:Bombesin