Abstract

G17-Gly has been shown to stimulate the growth of DLD-1 human colon cancer cells in a biphasic manner via high and low affinity receptors. In the current study, the existence of heterogeneous receptor populations for G17-Gly on the HT-29 human colon cancer cell line was investigated. The effect of either N- or C-terminal peptide truncation on receptor binding and cell growth stimulation was also explored. [Leu¹⁵]G17-Gly bound to both high (nM) and low (μM) affinity sites on HT-29 cells. The peptide stimulated cell growth in a dose-dependent and biphasic manner with maximal stimulation at 10⁻⁹ M peptide concentration, suggesting that, as in the case of DLD-1 cells, it is the high affinity receptor which is responsible for the growth-promoting effects. In contrast, G17(1–12) stimulated the growth of HT-29 cells in a sigmoidal fashion with an EC₅₀ of 4.6 × 10⁻⁹ M. Sequential N-terminal truncation of [Leu¹⁵]G17-Gly results in decreased binding to the high affinity G17-Gly receptor on DLD-1 cells. [Leu¹⁵]G17(11–17)Gly bound to the low affinity G17-Gly receptor with an affinity similar to that of the full sequence peptide but was unable to displace the radioligand from high affinity sites. G17(1–6)-NH₂ was unable to displace [³H]G17-Gly from either site. These results suggest that the important residues for binding to the low affinity receptor are in the C-terminal region of the peptide while those required for interaction with the high affinity receptor lie further towards the N-terminus.

Keywords: Glycine-extended gastrin; Colon cancer; Biphasic growth response; High affinity receptor; Low affinity receptor; Gastrin fragment

Abbreviations: BSA, bovine serum albumin; CCK-8, cholecystokinin-8; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; ESI-MS, electrospray ionization mass spectrometry; FCS, fetal calf serum; Fmoc, 9-fluorenylmethyloxycarbonyl; G17, gastrin; G17-Gly, N-carboxymethyl gastrin; [³H]G17-Gly, [3′,5′-3H-Tyr12,Leu15]G17-Gly; HBTU, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; THF, tetrahydrofuran; TFA, trifluoroacetic acid; Tris–HCl, tris hydroxymethyl aminomethane hydrochloride

Article Outline

1. Introduction

2. Materials and methods

2.1. Cells and reagents

2.2. Peptide synthesis

2.3. Peptide purification and characterization

2.4. Production of radiolabeled [Leu¹⁵]G17-Gly

2.5. Radioligand binding

2.6. Cell growth

2.7. Statistical analysis

3. Results

3.1. Binding of [Leu¹⁵]G17-Gly to HT-29 membranes and whole cells

3.2. Binding of [Leu¹⁵]G17-Gly analogs to DLD-1 membranes

3.3. Mitogenic effects of [Leu¹⁵]G17-Gly and G17(1–12) on HT-29 cells

4. Discussion

Acknowledgements

References