The aim of the present research is to determine the mechanism by which receptors of the gastrin family of peptides confer a growth advantage on gastrointestinal malignancies. The experiments will determine whether conferred growth is a result of normal receptor ligand binding or a result of abnormalities of the gastrin receptor. We will determine the role of receptor expression regulation, such as up or down regulation and internalization, as well as the role of abnormally expressed receptors, the role of receptors specific for ligands other than gastrin, whether there is a functional uncoupling of receptor binding from receptor expression, or whether a combination of these exist and accounts for the growth stimulation of gastrointestinal tumors.
The specific aims are as follows: 1) Determine the expression and the dynamics of expression of gastrin receptors on human colon and gastric cancer cells. We will determine the expression of gastrin receptor transcript and protein. This will be correlated with peptide binding studies to determine whether a functional uncoupling exists. This will be accomplished by Northern analysis, TR-PCR, antibody immunofluorescence, ligand binding, and Southern analysis. Cultured cells and surgical specimens will be utilized. 2) Determine the mitogenic effect of the gastrin receptor on human colon and gastric cancer cells. Cells bearing receptors which maintain a normal binding and expression relationship will be characterized by proliferation assays using specific receptor agonists and antagonists. Cells bearing receptors which do not exhibit a receptor binding-expression relationship will be studied in proliferation assays using anti-sense constructs. 3) Demonstrate the interaction of gastrointestinal peptides with gastrin and the gastrin receptor on human colon and gastric cancer cells. Peptide and receptors other than those of the gastrin family will be analyzed for a possible effect on the dynamics of gastrin receptor expression. This will be accomplished using biochemical and molecular probes for the bombesin, VIP, and muscarinic cholinergic families of peptide and neurotransmitters. Determinations will be performed by Northern and Southern analysis, and radiolabelled ligand binding.
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