Autocrine action of amphiregulin in a colon carcinoma cell line and immunocytochemical localization of amphiregulin in human colon

Abstract

Amphiregulin (AR) is a newly discovered glycosylated, 84-amino acid residue polypeptide growth regulator which has sequence homology to the EGF family of proteins. To obtain immunological reagents to study the biological role of AR, two synthetic peptides containing sequences corresponding to distinct regions of AR were used to generate polyclonal antibodies in rabbits. One preparation of antipeptide antibodies directed against residues 26-44 of AR (AR-Ab2) was most effective in the detection of native AR, whereas another preparation of antibodies against residues 8-26 (AR-Ab1) was found to be most efficacious in the detection of AR in formalin-fixed and paraffin-embedded tissues. The growth of a colon carcinoma cell line, Geo, which proliferates autonomously under serum-free conditions, was stimulated by the exogenous addition of AR or EGF. Half-maximal stimulation of this growth was observed at 40 and 200 pM of EGF and AR, respectively. A mAb to the extracellular domain of the EGF receptor blocked the stimulation of cell proliferation induced by the exogenous addition of AR, suggesting that this stimulation was mediated via the EGF receptor. Geo cells were found to constitutively express significant levels of the AR mRNA transcript as determined by analysis of the polymerase chain reaction-amplified cDNA product and AR protein was detected immunocytochemically using the AR-Ab1 antibodies in these cells. AR was immunoprecipitated specifically using the AR-Ab2 antibodies from the conditioned medium of Geo cells, which had been metabolically labeled with [35S]cysteine. The secreted AR migrated as a broad band (18.5-22.5 kD) with a median molecular weight of ∼20.7 kD in SDS-PAGE. Immunospecific removal of AR from serumfree medium conditioned by the Geo cells and readdition of the AR-depleted medium to Geo cells resulted in an ∼40% inhibition of cell growth relative to controls. Furthermore, the growth of the Geo cells was also inhibited by ∼50% by the addition of the anti-EGF receptor mAb alone. These results indicate that AR and the EGF receptor are involved in the autocrine growth of these cells and suggests that AR may act through the EGF receptor via an extracellular autocrine loop. To study the expression of AR in human colon in vivo, AR was localized immunocytochemically in formalin-fixed, paraffin-embedded sections from normal and malignant human colon using the AR-Ab1 antibodies. In all normal colon specimens, AR was localized to the cytoplasm and nucleus of the terminally differentiated, non-proliferative surface columnar and secretory epithelial cells of the mucosa, but was not detectable in the proliferative epithelial cells of the crypts. In four out of five colonic carcinomas, AR was localized to the cytoplasm and/or nucleus of the cells. In conclusion, AR can act as an autocrine growth stimulator for colonic carcinoma cells in vitro and may perform a growth regulatory function in normal and malignant colon in vivo.