Autocrine stimulation of growth of AR4-2J rat pancreatic tumour cells by gastrin

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Abstract
The control of cell proliferation by gastrin has been investigated in a rat pancreatic tumour cell line, AR4-2J. Exogenous gastrin, 10(-12) to 10(-8) M, stimulated cell growth of thymidine-synchronised AR4-2J cells cultured over 48 h in serum-free medium. Cell lysates of AR4-2J cells contained an average of 4.5 and 3.5 pg gastrin per 10(6) cells, when grown in serum-supplemented or serum-free media, respectively, as revealed by radioimmunoassay. In serum-free medium, AR4-2J secrete 34 ng 1(-1) 10(-6) cells of gastrin over 48 h. Addition of an anti-gastrin immunoglobulin preparation, but not control immunoglobulins, caused a maximum 52% reduction in cell growth. These data are consistent with an autocrine role for gastrin in the control of AR4-2J cell growth. These results were supported by studies with gastrin/CCK receptor antagonists. Six non-peptide gastrin/CCK receptor antagonists inhibited AR4-2J cell growth in a concentration-related manner. The concentration required for 50% inhibition (IC50) of cell growth by the amino acid-derived antagonists proglumide (3.5 x 10(-3) M), benzotript (1.8 x 10(-3) M), loxiglumide (1.1 x 10(-4) M) and lorglumide (6.7 x 10(-5) M) were of the same order and significantly correlated with their IC50 for inhibition of 125I-gastrin binding to AR4-2J cells. Inhibition of cell growth by these antagonists was partially reversed by the addition of exogenous gastrin. In contrast, the IC50 for inhibition of cell growth with two benzodiazepine-derived antagonists, the CCK-B receptor antagonist L-365,260 (4.6 x 10(-5) M) and the CCK-A receptor antagonist devazepide (1.7 x 10(-5) M) were two-three orders of magnitude greater than those required to inhibit gastrin binding (10(-8)-10(-7) M). The growth inhibitory effects of L-365,260 and devazepide were not reversed by exogenous gastrin suggesting these benzodiazepine-derived antagonists do not inhibit cell growth by interaction with gastrin receptors. The results are consistent with gastrin being an autocrine growth factor in AR4-2J cells, and that stimulation of cell growth is due to stimulation of the gastrin, rather than CCK-B, receptor sub-type. This study highlights that gastrin receptor antagonists warrant further investigation as agents to control growth of tumours, such as those from the gastrointestinal tract, which express gastrin receptors.