NSC-737664 The results sug gested that while neurotensin acted predominantly through PKC in Panc 1 cells and via EGFR transactiva tion in HT29 cells, it used both these pathways Inhibitors,Modulators,Libraries in HCT116 cells. In the latter cells neurotensin induced activation of ERK was mediated largely by PKC, while neurotensin induced activation of Akt was independent of PKC but involved transactivation of the EGFR, appar ently by a Ca2 dependent mechanism. Neurotensin induced DNA synthesis was mediated mainly by PKC. Methods Chemicals Dulbeccos modified Eagles medium, N piperazine N. penicillin and streptomycin were from Gibco. Neurotensin, 12 O tetradecanoylphorbol Inhibitors,Modulators,Libraries 13 acetate, thapsigargin, epidermal growth factor, and wortmannin were obtained from Sigma Aldrich.

maleimide, 4 6,7 dimethoxyquinazoline, 2 amino 3 methoxyflavone 2 4 methylpentanoyl Inhibitors,Modulators,Libraries L tryptophan methylamide were from Calbiochem. 7 Methyl 2 9 4H pyrido pyrimidin 4 one was obtained from Cayman Chemical. Transforming growth factor a was obtained from Bachem. 4 Qui nazolinamine, N 7 methoxy 6 was a gift from Astra Zeneca, and cetuximab was kindly provided by Merck KgaA. thymidine and myo inositol were from Amersham Biosciences. Antibodies against phosphory lated AktSer473, total Akt, dually phosphorylated ERKThr202Tyr204, phospho EGF receptorTyr1173, and phospho Shc Tyr239240 were obtained from Cell Signal ing Technology. Anti ERK and anti Shc antibodies Inhibitors,Modulators,Libraries were obtained from Upstate. EGFR antibody was obtained from Santa Cruz Biotechnology, Inc. Secondary antibo dies were purchased from Bio Rad Laboratories and Licor Biosciences.

All other chemicals were of analytical quality. Stock solu tions of test compounds were prepared in DMSO or 0. 9% NaCl. EGF was dissolved in 4 mM HCl, and TGFa in 4 mM HCl containing 1% bovine serum albumin from Sigma Aldrich. Cetuximab was dissolved in phosphate buffered saline. When solutions con taining DMSO were used, Inhibitors,Modulators,Libraries the final concentration of DMSO was kept as Nutlin-3a molecular weight low as possible. Cell culture Human colorectal cancer cell lines HCT116 and HT29, and pancreatic adenocarcinoma cell line Panc 1 were obtained from ATCC. The cells were maintained in Dulbeccos modified Eagles medium con taining 1 gl glucose supplemen ted with 10% horse serum, penicillin, streptomycin and 2 mM glutamine. Cells were plated onto Costar plastic culture wells at a density of 50 000 cells cm2 in serum containing medium. The cultures were kept in 95% air5% CO2 at 37 C. After 24 hours the medium was replaced with serum free medium and the cells were cultured for 24 hours before stimulation with agonists. Measurement of DNA synthesis Neurotensin, TPA, and inhibitors of PKC and EGF receptor were added to serum starved HCT116 cells as described in the figure legends, and thymidine was added 12 hours after stimulation.