Rogers B E, Rosenfeld M E, Khazaeli M B, Mikheeva G, Stackhouse M A, Liu T, Curiel D T, Buchsbaum D J
Department of Radiation Oncology, University of Alabama at Birmingham 35294, USA.
J Nucl Med. 1997 Aug;38(8):1221-9.
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.
[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.
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.
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.
胃泌素释放肽受体（GRPr）对14个氨基酸的蛙皮素肽具有高亲和力。在此分析中，将[125I]-Tyr4-蛙皮素与[125I]-mIP-蛙皮素（一种7个氨基酸的蛙皮素类似物）进行比较，用于体外结合、内化进入肿瘤细胞以及体内肿瘤定位。此外，使用重组腺病毒载体（AdCMVGRPr）进行基因转移，以诱导人卵巢癌细胞中GRPr的表达，用于与这些放射性标记肽的结合和肿瘤定位。
合成[125I]-mIP-蛙皮素，并在24小时内使用BNR-11细胞（稳定转染GRPr的小鼠成纤维细胞）在内化试验中与[125I]-Tyr4-蛙皮素进行比较。体外结合试验使用BNR-11、A427、HeLa和SKOV3.ip1人癌细胞，这些细胞要么未感染，要么感染了AdCMVGRPr。在正常BALB/c小鼠和携带原位SKOV3.ip1卵巢癌肿瘤的无胸腺裸鼠中进行生物分布研究。用AdCMVGRPr腺病毒载体诱导SKOV3.ip1肿瘤表达GRPr。
内化试验表明，[125I]-Tyr4-蛙皮素在37℃下迅速内化并分解代谢，4小时时约10%的放射性保留在细胞内，而[125I]-mIP-蛙皮素约为30%。HeLa、A427和SKOV3.ip1细胞均被诱导表达高于阳性对照BNR-11细胞的GRPr水平。正常小鼠在注射后4小时，[125I]-mIP-蛙皮素在血液和甲状腺中的放射性水平[每克注射剂量的0.26%±0.10%（ID/g）和0.24%±0.05% ID]低于[125I]-Tyr4-蛙皮素（3.5%±1.6% ID/g和5.2%±4.4% ID）。接种肿瘤细胞后5天腹腔内（i.p.）给予AdCMVGRPr，然后在第7天腹腔内给予[125I]-mIP-蛙皮素的携带i.p. SKOV3.ip1肿瘤的小鼠，在注射后4小时肿瘤中的放射性为16.5%±4.8% ID/g，而[125I]-Tyr4-蛙皮素为5.9%±3.0% ID/g。给予盐水或表达β-半乳糖苷酶（LacZ）基因的对照腺病毒的荷瘤小鼠，两种蛙皮素肽的肿瘤摄取值均显著较低。
内化试验表明，与[125I]-Tyr4-蛙皮素相比，[125I]-mIP-蛙皮素在细胞内化和保留方面具有良好的特性。结果证明用表达GRPr的重组腺病毒载体成功地在体外和体内转导人肿瘤细胞。此外，与[125I]-Tyr4-蛙皮素相比，体内转导以表达GRPr的肿瘤显示出[125I]-mIP-蛙皮素的定位显著更高。
