For example, overexpression of the EGFR agonist transforming grow

For example, overexpression of the EGFR agonist transforming growth factor alpha (TGF��) in mice causes hepatic hyperplasia and tumour Ponatinib molecular weight formation [21,22], and EGFR is upregulated in a majority of human hepatocarcinomas [23]. Inhibition of the EGFR by antibodies or tyrosine kinase blockers can attenuate the growth of hepatocarcinoma cells in vitro [24,25], and are currently being tested in clinical trials in hepatocarcinomas [26]. Prostaglandins, acting through different receptors of the GPCR family, regulate many cellular functions [27]. In epithelial cells, prostaglandins often enhance proliferation and survival, and several lines of evidence implicate them in oncogenesis [28]. In many tumours, cyclooxygenases (COX-1 and COX-2), which catalyze the rate-limiting step in prostaglandin synthesis, are overexpressed, and the levels of prostaglandins, notably prostaglandin E2 (PGE2), are elevated [28-31].

In hepatocytes, PGE2 and other prostaglandins enhance DNA synthesis [15,32-34], and COX-2 is overexpressed in many hepatocarcinomas [35,36]. In the study presented here we examined the Morris hepatocarcinoma cell line MH1C1, which was chosen due to its responsiveness to both EGF and the prostaglandins PGE2 and PGF2��, and investigated the interaction between the pathways mediated by prostaglandin receptors and EGFR. We previously observed that while there was no evidence of transactivation of EGFR induced by prostaglandins or other GPCR agonists in hepatocytes, PGE2 induced phosphorylation of the EGFR in the MH1C1 cells [37,38]. We have now investigated further the signalling mechanisms involved in this effect.

Methods Chemicals Dulbecco��s Modified Eagle’s Medium, Dulbecco��s phosphate-buffered saline, William��s Medium E, glutamine, and Pen-Strep (10.000 U/ml) were from Lonza(Verviers, Belgium). HEPES was from Gibco (Grand Island, NY). Dexamethasone, insulin, bovine serum albumin, collagen (type I, rat tail), prostaglandin F2�� (Tris salt) and epidermal growth factor (EGF) were obtained from Sigma-Aldrich (St.Louis, MO). GF109203X ([2-[1-(3-dimetylaminopropyl)-1H-indol-3-yl]-male-imide]) and GM6001/Galardin (N-[(2R)-2 (hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) were from Calbiochem (San Diego, CA). Gefitinib was a gift from AstraZeneca (Cheshire, UK). [6-3H]thymidine (20�C30Ci/mmol) and myo-[2-3H]inositol (15.

0Ci/mmol) were from PerkinElmer (Boston, MA). AL8810 (9��,15R-dihydroxy-11��-fluoro-15-(2,3-dihydro-1H-inden-2-yl)-16,17,18,19,20-pentanor-prosta-5Z,13E-dien-1-oic acid),L161982 (N-[[4'-[[3-butyl-1,5-dihydro-5-oxo-1-[2-(trifluoromethyl)phenyl]-4H-1,2,4-triazol-4-yl]methyl][1,1'-biphenyl]-2-yl]sulfonyl]-3-methyl-2-thiophenecarboxamide), GSK-3 (+)fluprostenol, and prostaglandin E2 (PGE2) were from Cayman Chemical (Ann Arbor, MI).

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