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Specific Binding and Growth Effects of Bombesin-related Peptides on Mouse Colon Cancer Cells in Vitro¹

Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77550

In the present study, we characterized specific binding of bombesin (BBS)/gastrin-releasing peptide (GRP) to mouse colon cancer (MC-26) cells. MC-26 cells were inoculated into male BALB/c mice subdermally, and tumors were harvested from mice 21–28 days postinoculation. Tumor membranes were analyzed for binding to GRP-related peptides, using either ¹²⁵I-GRP or ¹²⁵I-tyrosine⁴-BBS. Under optimal binding assay conditions, BBS displaced specific binding of both ¹²⁵I-GRP and ¹²⁵I-tyrosine⁴-BBS in a dose-dependent manner, and a curvilinear displacement resulted. Specific binding data, analyzed by either a Scatchard or a Lineweaver-Burk plot, demonstrated presence of 2 classes of specific binding sites, arbitrarily named type I and type II sites. Type I sites had a high binding affinity [K_d 0.45 ± 0.05 nM (SE)] and a relatively low capacity (226 ± 27 fmol/mg membrane protein), whereas type II sites had a 10–20-fold lower binding affinity and 6–7-fold higher capacity. BBS/GRP binding sites were specific for GRP-related peptides and demonstrated no significant binding affinity for all other unrelated peptides tested. Relative binding affinity of GRP analogues was in the order of GRP (14–27) > neuromedin C BBS GRP (1–27) > neuromedin B (for the later, P > 0.05 versus other peptides). Two BBS receptor antagonists, [D-Arg¹,D-trp^7,9,Leu¹¹]-substance P (spantide) and [Leu¹³--(CH₂NH)Leu¹⁴]BBS also inhibited specific binding of ¹²⁵I-GRP in a dose-dependent manner. Molecular weight of GRP/BBS binding proteins on tumor membranes was determined by cross-linking methods. A major molecular form (>80–90%) (M_r 75,000) and a minor M_r 180,000 band were evident, both under reducing and nonreducing conditions. BBS (0.5–50 nM) demonstrated a significant dose-dependent growth effect on MC-26 cells in vitro, in terms of [³H]thymidine and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptake; these studies indicate that the BBS/GRP binding sites on MC-26 cells may serve as functional receptors and mediate the growth effects of BBS on MC-26 cells.

¹ This study was supported by NIH Grants DK 35608 and CA 38651, and American Cancer Society Grant PDT-220.

² To whom requests for reprints should be addressed, at University of Texas Medical Branch, Department of Surgery, 6.202 Old John Sealy, Route E32, Galveston, TX 77550.

Received 5/ 7/90. Accepted 7/24/90.

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