EXPERIMENTAL CELL RESEARCH 223, 268–273 (1996) ARTICLE NO. 0081 Autocrine Growth Loop of The Epidermal Growth Factor Receptor in Normal and Immortalized Human Bronchial Epithelial Cells MING-SOUND TSAO,* , † ,1 HONG ZHU,† AND JEAN VIALLET‡ *Department of Pathology, Montreal General Hospital/Research Institute and †McGill University, Montreal, Quebec, Canada H3G 1A4; ‡Centre d’oncologie, Ho ˆpital Notre Dame, 1560 est, Rue Sherbrooke, Montreal, Quebec, Canada H2L 4M1 pression of transforming growth factor-a (TGF-a) and Epidermal growth factor (EGF) is a potent growth its receptor, the epidermal growth factor receptor factor for human normal bronchial epithelial (HBE) (EGFR) in human lung cancer cell lines and tissue, cells and lung cancer cells, which often demonstrate especially the non-small cell lung carcinomas (NSCLC), an EGF receptor (EGFR) autocrine loop. We have thus supporting the concept that a TGF-a/EGFR auto- found that HBE cells are capable of proliferating in crine loop plays an important role in the pathogenesis basal medium without EGF supplementation, and this and biology of human lung cancer [6–14]. In fact, the suggests the probable presence of an active EGFR au- functional activity of the putative autocrine growth tocrine loop in non-neoplastic HBE cells. Northern blot loop involving TGF-a and EGFR has been demon- hybridization shows that the parental and immortal- strated in several lung carcinoma cell lines by the inhi- ized HBE cells express comparable and high levels of bition of their proliferation after the addition of a neu- mRNA for EGFR, transforming growth factor-alpha tralizing monoclonal antibody against TGF-a to their (TGF-a), and amphiregulin (AR), but not EGF. Incuba- culture medium [8, 14]. tion with neutralizing monoclonal antibodies (mAb) Normal human bronchial epithelial (HBE) cells have against EGFR partially inhibits the growth of these been successfully propagated in monolayer culture us- cells. Immunohistochemistry shows that HBE cells ex- ing serum-free culture media which contain growth press the TGF-a peptide in vitro and in vivo, however, supplements, including cholera toxin (CT), epidermal neutralizing mAbs against TGF-a fail to inhibit their growth factor (EGF), and bovine pituitary extract proliferation. In contrast, AR stimulates the growth of (BPE) [15, 16]. We and others have reported that the HBE cells. Thus, several EGF-family ligands appear to transfection and expression of the SV40 large T antigen be involved functionally in the EGFR autocrine [17] or the E6/E7 gene of the human papilloma virus growth loop in HBE cells.  1996 Academic Press, Inc. [18, 19] are capable of immortalizing these HBE cells. We report here that cultured HBE cells and their im- mortalized lines coexpress EGFR, TGF-a, and amphi- INTRODUCTION regulin (AR), another member of the EGF-family growth factors [20, 21], and that this growth factor loop Most cell functions in multicellular organisms are represents an important autocrine regulator for the regulated by polypeptide growth factors through inter- proliferation of HBE cells, predating their neoplastic actions with their specific receptors [1 – 3]. An autocrine transformation. growth control loop is putatively present when both the receptor and its binding ligand are coexpressed in a MATERIALS AND METHODS single cell, thus providing an opportunity for such a cell to regulate its own proliferation. The ‘‘autocrine Keratinocyte serum-free medium, Ca 2/ and Mg 2/ -free Hanks’ bal- anced salt (HBSS) and typsin-EDTA solutions were purchased from growth loop’’ model was first formulated to suggest a Gibco-BRL (Grand Island, NY). Neutralizing mouse monoclonal anti- mechanism for neoplastic cells to escape the normal bodies LA1 (against EGFR, Catalog No. 05-101) and hEGF-H homeostatic regulation of cell growth and expand clon- (against human EGF, Catalog No. 05-109) were purchased from UBI ally during promotion and progression in carcinogene- (Lake Placid, NY). Monoclonal antibodies 528 (against EGFR) and sis [4, 5]. Many investigators have reported the coex- 189 (against TGF-a) were obtained from Santa Cruz Biotech (Santa Cruz, Ca). Monoclonal antibodies Ab-3 (neutralizing) and Ab-2 (im- muno-histochemistry) against TGF-a were purchased from Oncogene 1 To whom reprint requests should be addressed at present ad- Science (Cambridge, MA). All the monoclonal antibodies were of mouse origin and represent affinity-purified immunoglobulins (IgG). dress: Department of Pathology, Ontario Cancer Institute/Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Can- AR was purchased from R&D (Minneapolis, MN) and TGF-a from Bachem (Torrance, CA). ada M5G 2M9. 268 0014-4827/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.