Ensayos

Published August 1, 1988

Two Independent Mechanisms for Escaping Epidermal Growth Factormediated Growth Inhibition in Epidermal Growth Factor Receptor-hyperproducing Human Tumor Cells
M a s a m i c h i Hirai,* S h i n o b u G a m o u , * Shinsei Minoshima,* a n d N o b u y o s h i Shimizu** * Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku,Tokyo 160, Japan; and *Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721

Abstract. Human squamous cell carcinoma cell lines
often possess increased levels of epidermal growth factor (EGF) receptor. The growth of these EGF receptor-hyperproducing cells is usually inhibited by EGE To investigate the mechanism of EGF-mediated inhibition of cell growth,variants displaying alternate responses to EGF were isolated from two squamous cell carcinoma lines, NA and Ca9-22; these cell lines possess high numbers of the EGF receptor and an amplified EGF receptor (EGFR) gene. The variants were isolated from NA cells after several cycles of EGF treatment and they have acquired EGF-dependent growth. Scatchard plot analysis revealed a decreased level of EGFreceptor in these ER variants as compared with parental NA cells. Southern blot analysis and RNA dot blot analysis demonstrated that the ER variants had lost the amplified EGFR gene. One variant

isolated from Ca9-22 cells, CER-1, grew without being affected by EGE CER-1 cells had higher numbers of EGF receptor than parental Ca9-22 but similar EGFR gene copy number. Flow cytometric analysis indicatedan increase in ploidy and cell volume which may give rise to the increase in receptor number per cell. The EGF receptors on both Ca9-22 and CER-1 cells were autophosphorylated upon EGF exposure in a similar manner suggesting no obvious alteration in receptor tyrosine kinase. However, very efficient down-regulation of the EGF receptor occurred in CER-1 cells. These data suggest two independentmechanisms by which EGF receptor-hyperproducing cells escape EGF-mediated growth inhibition: one mechanism is common and involves the loss of the amplified EGFR genes, and another is novel and involves the efficient down-regulation of the cell-surface receptor.

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PIDERMAL growth factor (EGF) 1 is a potent mitogen for a variety of cells (Carpenterand Cohen, 1979). The mitogenic signal of EGF is transduced through its membrane-anchored glycoprotein receptor of Mr 170,000 (Cohen et al., 1982). The binding of EGF to the receptor induces a series of biochemical reactions including autophosphorylation of the EGF receptor (Cohen et al., 1980), clustering of the EGF receptor into coated pits (Schechter et al., 1979) and internalization andintracellular processing of the EGF/EGF receptor complex (Schlessinger et al., 1978; HaiRier et al., 1978; Miskimins and Shimizu, 1984). Concurrently, cellular protein phosphorylation (Hunter and Cooper, 1981), c-fos and c-myc protooncogene expression (Mfiller et al., 1985) and phosphatidylinositol turnover (Sawyer and Cohen, 1981) are induced. However, the presence of EGF at the concentrationsmitogenic for other cells was found to be markedly inhibitory for the growth of the epidermoid carcinoma cell line A431 (Gill and Lazar, 1981; Barnes, 1982), which possesses extremely high numbers of the EGF receptor (Fabricant et al., 1977)
1. Abbreviations used in this paper: EGE epidermal growth factor; EGFR, epidermal growth factor receptor (genes).

as well as EGF receptor (EGFR) geneamplification (Lin et al., 1984; Ullrich et al., 1984). Similar growth inhibition has been reported for the breast cancer cell line MI3A-468 (Filmus et al., 1987), which also possesses high numbers of the EGF receptor. To determine which biochemical response is crucial for growth inhibition of these cells and also for growth stimulation of cells with ordinary numbers of EGF receptor, we and others have...