GRP-receptor-mediated signal transduction, gene expression and DNA synthesis in the human pancreatic adenocarcinoma cell line HPAF

Abstract

Bombesin-like peptides have been implicated as growth factors in various human cancers. Human adenocarcinoma cell lines (Capan-1, Capan-2, MiaPaCa-2 and HPAF) were tested to determine whether they express the gastrin-releasing peptide-preferring bombesin receptor (GRPR) and neuromedin B-preferring bombesin receptor (NMBR). Using RT-PCR the highest level of GRP receptor mRNA was found in HPAF cells. NMB receptor mRNA expression moderate in all cell lines investigated. We therefore selected the HPAF cell line to investigate whether bombesin treatment affects intracellular Ca 2+ ([Ca 2+] i), cAMP level, DNA synthesis as a measure of cell proliferation, and expression of three transcription factors: c-fos, c-myc and high mobility group protein IY (HMG-I(Y)). Bombesin administration led to an immediate increase in free intracellular Ca 2+ concentration ([Ca 2+] i) but did not change cAMP levels. The peptide also enhanced [ 3H]thymidine incorporation in HPAF cells (but not in the other cell lines), an effect that was concentration dependent, reaching 36 ± 5% stimulation over control values at 24 h with an EC 50 of 2.27 × 10 −12 M. Furthermore, bombesin stimulated c-fos, c-myc and HMG-I(Y) expression in a time-dependent manner: the c-fos mRNA level increased dramatically in the first 30 min of exposure, then returned to basal level within 2 h, while the c-myc and HMG-I(Y) mRNA levels peaked at 2 h and 4h, respectively. All actions of bombesin were blocked by BME (D-Phe 6-bombesin-(6–13)-methylester), a selective GRP receptor antagonist, but not by the NMB receptor antagonist BIM-23127 (D-Nal-cyclo[Cys-Tyr-D-Trp-Orn-Val-Cys]-Nal-NH 2). We conclude that HPAF cells express mRNA for GRP receptors and that functional receptors are present in the cell membrane. The occupation of these receptors leads to a sequence of intracellular events involving rapid mobilization of intracellular Ca 2+, expression of c-fos, c-myc and HMG-I(Y) mRNA, and stimulation of cell proliferation. Conversely, although NMB receptor mRNA can be detected, its actual translation to functional receptors does not reach a detectable level.