Pergamon zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Archs oral Bid. Vol. zyxwvutsrqponmlkjihgfe 39, No. 7. 569-580, 1994 pp. Copyright0 1994 Elsevier Scmm Ltd Printed in Great Britain. All rightsreserved 0003-9969/94 $7.00 + 0.00 zyxwvu CO-EXPRESSION OF EPIDERM A L GROWTH FACTOR-RECEPTOR AND c-erb B-2 PROTO-ONCOGENE PRODUCT IN HUMAN SALIVARY-GLAND ADENOCARCINOMA CELL LINE HSG AND THE IMPLICATIONS FOR HSG CELL AUTOCRINE GROWTH S. KYAKUMOTO, R. KUROKAWA, M. HOSHINO and M. OTA Department of Biochemistry, Iwate Medical University School of Dentistry, Morioka 020, Japan (Accepted 22 February 1994) Summary-The autonomous proliferation of HSG cells is mediated by an autocrine growth factor, a 46K epidermal growth factor (EGF)-like molecule. The receptor for this molecule was investigated. Immuno- precipitation and immunoblotting revealed the expression of two possible receptor molecules, EGF-R and p185 r’bB2, in HSG cells. Northern blotting also revealed the co-expression of 5.6-kb EGF-R mRNA and 4.6-kb c-erbB-2 mRNA. When the purified EGF-like molecule was added to the cultures, EGF-R but not ~185”‘~a-~ was autophosphorylated. These results suggest that, although both EGF-R and ~185”~“~ are co-expressed in HSG cells, the EGF-R is the genuine receptor for the EGF-like molecule. However, there is a possibility that p185”‘bB-2 IS Involved in the signal transduction system. This possibility was examined by using specific antibodies to human EGF-R (hEGF-R), p185”*“‘, and EGF to inhibit the functions of these molecules. Addition of these three antibodies to the cultures inhibited the growth of HSG cells. The antibodies to EGF-R and ~185 rrbs* also caused morphological changes such as disturbances of the plasma membrane, and some cell death. Surprisingly, the effect of the anti-p185”bs-2 antibody on growth inhibition and morphology was stronger than that of the anti-hEGF-R antibody. Thus, ~185”~~-~ expressed in HSG cells has an important function in the signal transduction of HSG cell growth. Key words: EGF receptor, c-erbB-2, p185Y’bs-*, salivary gland tumour, high molecular-weight EGF, (human). INTRODUCTION The human zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA c- erb B- 1 and c- erb B-2 proto-oncogenes are known to encode homologous membrane receptors, a IV, 170K EGF-R, pl 70”‘*B-“EGF-R (Downward et aI., 1984) and a M, 185K c-erbB-2 protein, p185”bB-2, that is also a transmembrane glycoprotein with intrinsic tyrosine kinase activity like the EGF-R (Akiyama et al., 1986). Because of the structural similarity between p185”hB-2 and EGF-R, ~185”~~-’ was also thought to be a growth-factor receptor (Yamamoto et al., 1986a); however, the ligand for ~185”“~*’ had not been fully characterized until quite recently. The normal ligands for EGF-R, EGF and transforming growth factor-a (Derynck, 1988) do not interact directly with ~185”~‘~~ (Stern and Kamps, 1988; King et al., 1988; Kokai et al., 1988; Connelly and Stern, 1990). However, a IV, 30K protein (gp30), which is secreted from the MDA-MB-231 human Abbreviations: EGF-R, epidermal growth factor-receptor; hEGF, human epidermal growth factor; p185”ba-*, c-erbB-2 proto-oncogene product; MEM, Eagle’s mini- mal essential medium; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SSC, standard sodium citrate. breast-cancer cell line and interacts directly with EGF-R and ~185 erbB-2 is reportedly a ligand for ~185”“~-~ (Lupu et al., 1990). Recently, Holmes et al. (1992) purified a M, 45K protein, heregulin-a, from conditioned medium of the same cell line and identified cDNA clones encoding related heregulins, all of which are similar to proteins in the EGF family. We previously identified an autocrine growth fac- tor, an EGF-like molecule having a high molecular weight (M, 46K) and recognized by anti-hEGF anti- body, in the human salivary-gland adenocarcinoma cell line HSG (Kurokawa, Kyakumoto and Ota, 1989). Using anti-hEGF antibody-coupled Sepharose CL-4B column chromatography, we followed the purification of the molecule from medium con- ditioned by a subclone of HSG cells (HSG-SF) conditioned to grow in a serum-free, defined medium. The M, 46K EGF-like molecule thus obtained stimu- lated the growth of HSG cells dose-dependently, demonstrating the presence of an autocrine growth mechanism in HSG cells. Thus, the HSG cell culture provides a suitable model system for studying the autonomous growth of cancers in the human oral region. Moreover, the M, 46K EGF-like molecule may possibly be a marker for some human salivary-gland cancers.