The results sug gested that although neurotensin acted predominantly by means of PKC in Panc 1 cells and by means of EGFR transactiva tion in HT29 cells, it used both these pathways in HCT116 cells. From the latter cells neurotensin induced activation of ERK was mediated largely by PKC, whereas neurotensin induced activation of Akt was independent of PKC but concerned transactivation from the EGFR, appar ently by a Ca2 dependent mechanism. Neurotensin induced DNA synthesis was mediated primarily by PKC.
Strategies Chemicals Dulbeccos modified Eagles medium, N piperazine N penicillin and streptomycin have been from Gibco Neurotensin, 12 O tetradecanoylphorbol 13 acetate thapsigargin, epidermal development element and wortmannin selleckchem were obtained from Sigma Aldrich maleimide 4 six,7 dimethoxyquinazoline 2 amino three methoxyflavone 2 4 methylpentanoyl Rhein L tryptophan methylamide had been from Calbiochem seven Methyl two 9 4H pyrido pyrimidin 4 a single was obtained from Cayman Chemical Transforming development aspect a was obtained from Bachem four Qui nazolinamine, N 7 methoxy six was a gift from Astra Zeneca and cetuximab was kindly offered by Merck KgaA thymidine and myo inositol were from Amersham Biosciences Antibodies against phosphory lated AktSer473, total Akt, dually phosphorylated ERKThr202 Tyr204, phospho EGF receptorTyr1173, and phospho Shc Tyr239 240 had been obtained from Cell Signal ing Technologies Anti ERK and anti Shc antibodies have been obtained from Upstate EGFR antibody was obtained from Santa Cruz Biotechnology, Inc. Secondary antibo dies had been bought from Bio Rad Laboratories and Licor Biosciences All other chemical compounds have been of analytical superior. Stock solu tions of test lbs had been ready in DMSO or 0. 9% NaCl EGF was dissolved in four 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 have been made use of, the last concentration of DMSO was stored as very low as possible.