Protein kinase Calpha is implicated in cholecystokinin-induced activation of 70-kd S6 kinase in AR42J cells.

OBJECTIVES: We showed previously that cholecystokinin (CCK)-induced 70-kd S6 kinase activation is partly mediated by protein kinase C (PKC) in pancreatic acinar AR42J cells. Here, we examined which isoform of PKC is involved in this process. METHODS: AR42J cells were infected with adenovirus vectors carrying the kinase-deficient alpha, delta, and epsilon isoforms of PKC, the dominant-negative form of the 85-kd regulatory subunit of phosphatidylinositol (PI) 3-kinase, and the dominant-negative form of Sos. CCK-induced p70 S6 kinase activation was determined in AR42J cells infected with these adenovirus vectors. RESULTS: CCK-induced p70 S6 kinase activity was significantly reduced in cells overexpressing the dominant-negative p85 subunit of PI 3-kinase but not in cells overexpressing dominant-negative Sos or beta-galactosidase. CCK-induced p70 S6 kinase activity was inhibited in parallel with the expression levels of kinase-deficient PKCalpha, whereas it was unaffected by the expression of kinase-deficient PKCdelta or PKCepsilon. CONCLUSION: PKCalpha is implicated in CCK-induced activation of p70 S6 kinase in AR42J cells.

Requirement of autophosphorylated tyrosine 992 of EGF receptor and its docking protein phospholipase C gamma 1 for membrane ruffle formation.

Stimulation of the epidermal growth factor receptor (EGFR) produces membrane ruffles through the small G protein Rac1; however, the signaling pathway from EGFR to Rac1 has not yet been clarified. Here, we show that autophosphorylation of EGFR at tyrosine 992 is required for EGF-induced membrane ruffle formation in CHO cells. Signaling from the autophosphorylated tyrosine 992 appears to be mediated by phospholipase C (PLC) gamma 1. Furthermore, activation of Rac1 by EGF is inhibited by a PLC inhibitor. These results, taken together, suggest that autophosphorylation of EGFR at tyrosine 992 and the subsequent PLC gamma 1 activation transduce the signal to Rac1 to induce membrane ruffle formation.

Stimulatory effects of bilirubin on amylase release from isolated rat pancreatic acini.

Considered to be an etiologic factor of acute pancreatitis, hypersecretion of pancreatic juice and digestive enzymes is often associated with hyperbilirubinemia. We explored the intracellular mechanisms through which bilirubin affects pancreatic exocrine secretory function by examining the effect of bilirubin on isolated rat pancreatic acini. Bilirubin stimulated amylase release in a concentration- and time-dependent manner, significantly increasing amylase release at concentrations >5 mg/100 ml and after 15 min of incubation. Coincubation of bilirubin with vasoactive intestinal polypeptide, 8-bromo-cAMP, or A-23187 had a synergistic effect on amylase release, whereas coincubation with CCK-8, carbamylcholine, or 12-O-tetradecanoylphorbol 13-acetate had an additive effect. Bilirubin did not affect acinar cAMP content or Ca(2+) efflux. Intracellular Ca(2+) pool depletion had no influence on bilirubin-evoked amylase release. The protein kinase C (PKC) inhibitors staurosporine and calphostin C partially but significantly inhibited bilirubin-stimulated amylase release, whereas the PKA inhibitor H-89 did not. The tyrosine kinase (TK) inhibitor genistein, phospholipase A(2) (PLA(2)) inhibitor indoxam, and PLC inhibitor U-73122 also inhibited amylase release. Bilirubin significantly translocated PKC activity from the cytosol to the membrane fraction and activated TK in cytosol and membrane fractions. These results indicate that bilirubin stimulates amylase release by activating PKC and TK in rat pancreatic acini and that PLC and PLA(2) partly mediate this process.

Pedunculated early carcinoma of supra-ampullary duodenum presenting as acute pancreatitis.

Background. A 43-yr-old man was admitted to our hospital after sudden onset of epigastric pain. He was diagnosed with acute pancreatitis by clinical, laboratory, and radiographic signs. Examinations for the etiology of acute pancreatitis revealed a duodenal tumor arising at the proximal portion of the descending limb, extending by a long stalk, and coming into contact with Vater's papilla. The tumor was snare-resected endoscopically. Histological examination showed an early carcinoma. Extra-ampullary carcinoma of the duodenum should be considered an unusual cause of acute pancreatitis secondary to obstruction of the major duodenal papilla. Endoscopic polypectomy is effective because of the difficulty in making a precise diagnosis by endoscopic biopsy.

Role of Akt in growth and survival of PANC-1 pancreatic cancer cells.

INTRODUCTION: Akt is involved in different cellular processes such as cell growth, cell differentiation, and anti-apoptosis. AIMS: To investigate the role of Akt in cell growth and survival in PANC-1 pancreatic cancer cells. METHODOLOGY AND RESULTS: Insulin-like growth factor (IGF)-I induced Akt activation in a dose-dependent manner and stimulated anchorage-dependent and anchorage-independent cell growth of PANC-1 cells. In PANC-1 cells infected with adenovirus vectors carrying kinase-deficient Akt, anchorage-dependent and anchorage-independent cell growth was significantly reduced in the presence or absence of IGF-I compared with cells infected with adenovirus vectors carrying wild-type Akt, although IGF-I significantly stimulated cell growth in both transfected cell lines. Conversely, in PANC-1 cells infected with adenovirus vectors carrying kinase-deficient Akt, typical DNA laddering was undetectable in DNA fragmentation assay, and DNA 3;-OH reactivity was not detected in TUNEL assay. We then examined the role of phosphatidylinositol 3-kinase (PI3-K), an upstream mediator of Akt, on cell survival. In PANC-1 cells infected with adenovirus vector carrying a deletion mutant of the 85-kDa regulatory subunit of PI3-K and in cells treated with PI3-K inhibitor wortmannin, typical DNA laddering was evident in DNA fragmentation assay. In TUNEL assay, nuclear condensation and DNA 3;-OH reactivity was observed in approximately 30% of these cells. CONCLUSION: The present results indicate that Akt is implicated in cell growth, but not in survival in PANC-1 cells. These results suggest that there may be an alternative survival signal cascade from PI3-K in PANC-1 cells.

Shc and CEACAM1 interact to regulate the mitogenic action of insulin.

CEACAM1, a tumor suppressor (previously known as pp120), is a plasma membrane protein that undergoes phosphorylation on Tyr(488) in its cytoplasmic tail by the insulin receptor tyrosine kinase. Co-expression of CEACAM1 with insulin receptors decreased cell growth in response to insulin. Co-immunoprecipitation experiments in intact NIH 3T3 cells and glutathione S-transferase pull-down assays revealed that phosphorylated Tyr(488) in CEACAM1 binds to the SH2 domain of Shc, another substrate of the insulin receptor. Overexpressing Shc SH2 domain relieved endogenous Shc from binding to CEACAM1 and restored MAP kinase activity, growth of cells in response to insulin, and their colonization in soft agar. Thus, by binding to Shc, CEACAM1 sequesters this major coupler of Grb2 to the insulin receptor and down-regulates the Ras/MAP kinase mitogenesis pathway. Additionally, CEACAM1 binding to Shc enhances its ability to compete with IRS-1 for phosphorylation by the insulin receptor. This leads to a decrease in IRS-1 binding to phosphoinositide 3'-kinase and to the down-regulation of the phosphoinositide 3'-kinase/Akt pathway that mediates cell proliferation and survival. Thus, binding to Shc appears to constitute a major mechanism for the down-regulatory effect of CEACAM1 on cell proliferation.