Transforming growth factor-A-mediated growth pathways in human gastro-intestinal cell lines in relation to the gastrin autocrine pathway

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alp ha) increase transcription of the gastrin gene, and the gastrin peptide may be phosphorylated by EGF-stimulated tyrosine kinase. Our aims were to comp are EGF/TGF-alpha interactions in 2 human gastro-intestinal cell lines: MGL VAI, with a strong gastrin autocrine pathway, and C170HM2, with a weak path way. Both cell lines expressed the TGF-alpha gene. MGLVAI expressed TGF-alp ha protein as determined by immuno-cytochemistry, which was absent in C170H M2. Both cell lines expressed the same level of EGF receptors, as assessed by flow cytometry; however, MGLVAI did not have enhanced in vitro prolifera tion in response to EGF or TGF-alpha, unlike C170HM2. The basal growth of M GLVAI was inhibited by anti-sera against TGF-alpha, the EGF receptor and G1 7. C170HM2 was not inhibited by any of the anti-sera. Neutralisation of TGF -alpha resulted in undetectable cell-associated progastrin levels in MGLVAI (untreated had 391.7 fmol/5 x 10(6) cells), The progastrin level of C170HM 2 remained unaffected. Tyrosine kinase activity, as assessed by phosphopept ide concentration, of unstimulated MGLVAI was 2.6 times higher than that of C170HM2 in the cell membrane fraction (0.097 compared to 0.037 mu g/mg pro tein, p < 0.001) and 4.8 times higher in the cytosolic fraction (0.269 comp ared to 0.056 mu g/mg protein, p < 0.05). Following treatment with EGF, the phosphopeptide concentration increased in both the membrane and cytosolic fractions of both cell lines. Tyrphostin B42, which inhibits autophosphoryl ation of the EGF receptor, inhibited the basal growth of MGLVAI (IC50 1.3 m u M) and C170HM2 (9.5 mu M, p < 0.05 from MGLVAI), Herbimycin, which inhibi ts pp60(c-src) kinase, reduced the basal growth of MGLVAI (0.67 mu M) but n ot C170HM2. Immunofluorescence studies confirmed the presence of tyrosine-p hosphorylated proteins and pp60(c-src) within the cytoplasm of unstimulated MGLVAI cells. There was no specific immunofluorescence for either paramete r in C170HM2 cells until after treatment with EGF. Int. J. Cancer 87:20-28, 2000. (C) 2000 Wiley-Liss, Inc.