ICANÅ’RESEARCH 5.1.5548-5553. November is, tro) Loss of Thyrotropin Regulation and Transforming Growth Factor ß-induced Growth Arrest in erbft-2 Overexpressing Rat Thyroid Cells1 Gabriella Mincione, Anna Maria Cirafici, Davide Lazzareschi, Stefano Pepe, Fortunato Ciardiello, and Giulia Colletta2 Istituto di Patologia Umana e Medicina Sociale, Facoltà di Medicina e Chirurgia, Chieti (G. M., C. C./; Dipartimento di Medicina Sperimentale, Università La Sapienza, Rome [D. L.J; Centro di Oncologia ed Endocrinologia Sperimentale, Consiglio Nazionale delle Ricerche ¡A.M. C/; and Cattedra di Oncologia Medica /S. P., F. C.j, II Facoltà di Medicina e Chinirgia, Naples, Italy ABSTRACT Amplification of erbB-2 gene and overexpression of gpl85erb""2 gene product is found in approximately one-third of primary human breast and ovarian cancer. Overexpression of gpI85erh"~2 was recently found in hu man papillary thyroid carcinomas, but not in thyroid follicular carcino mas or adenomas. The erbB-2 gene encodes a cell surface growth factor receptor with intrinsic tyrosine kinase activity. Wild type human erbB-2 has been shown to act as a potent oncogene when overexpressed in mouse fibroblasts. To test whether overexpression of normal human erbB-2 gene can transform epithelial differentiated rat thyroid cells, these cells were infected with a recombinant retroviral expression vector containing the erbB-2 protooncogene. Rat thyroid cells expressing high levels of gpl85'rhB'2 do not display a fully transformed and tumorigenic phenotype. However, the isolated cell clones that over-express gpl85'pbB'2, show changes in their growth properties if compared to normal thyroid cells, since they can grow in absence of thyrotropin, the main growth factor controlling thyroid cell proliferation in vitro, and do not respond to the growth inhibitory effect of transforming growth factorß. INTRODUCTION Cell growth and differentiation are regulated in part by extracellular signals that are mediated by polypeptide molecules. Specific cell surface receptors that present oncogenic potential share a common catalytic function in their cytoplasmic domain, namely, a tyrosine- specific protein kinase activity (1). The erbB-2 oncogene encodes a tyrosine kinase (gpl85crhB~2), that is highly related to the receptor for the EOF3 (2, 3). Both gplSS"*10'2 and the EOF receptor share a cysteine-rich structure at their extracellular domains, which is con nected through a single transmembrane stretch of amino acids to a large cytoplasmic domain that carries the enzymatic function (4). Recently, the ligand of erbB-2 oncogene has been isolated, a Mr 44,000 glycoprotein named NDF (Neu differentiation factor) or he- regulin (5, 6). The oncogenic potential of this receptor can be released by multiple genetic mechanisms, including a point mutation within the transmem brane region and truncation of noncatalytic sequences at both the cytoplasmic and the extracellular domains (7-9). A third mechanism of oncogenic activation, which has been implicated in the pathogen- esis of human cancer, involves overexpression of the apparently nor mal gene (10, 11). Amplification and overexpression of erbB-2 have been detected at high frequency in human adenocarcinomas from several tissues. Overexpression of gpl85crhB"2 has been reported in breast and ovarian cancer and recently in thyroid papillary carcinoma (12). Overexpression of the human erbB-2 protooncogene leads to Received 3/10/93: accepted 9/13/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicale this fact. 1This work was supported by the progetto finalizzato ACRO Consiglio Nazionale delle Ricerche, and by the Associazione Italiana Ricerca sul Cancro. 2 To whom requests for reprints should he addressed 1The abbreviations used are: EGF, epidermal growth factor: TSH. thyrotropin; TGFß, transforming growth factor ß;MT-1, metallothionein-1: SDS-PAGE. sodium dodecyl sulfate-polyacrylamide gel clectrophoresis; cAMP. cyclic AMP. transformation of mouse NIH3T3 fibroblasts, while the oncogenicity of the homologous rat Neu protooncogene overexpressed in the same cells is less pronounced. The transformation process appears to be associated with constitutive tyrosine kinase activity and autophos- phorylation of the receptor molecule, in absence of ligand (8, 13, 14). Consistent with the hypothesis that this oncogene may also be involved in thyroid neoplasia, we tested its overexpression in an experimental model system to verify whether it leads to the appear ance of a transformed phenotype. Rat thyroid epithelial cells (FRTL-5 strain) represent a well char acterized system useful for studying the mechanisms of thyroid cell transformation by cellular protoncogenes and growth factors (15-17). This cell line has the peculiarity to retain in vitro the typical markers of thyroid differentiation such as thyroglobulin synthesis and secre tion, iodide uptake, and dependence on TSH for cell growth (18). When infected by viruses carrying the v-ki-ras, v-h-nzs, \-mos onco- genes, these cells lose their differentiated properties and acquire a fully transformed and tumorigenic phenotype (16, 17, 19). In the study presented here we infected FRTL-5 cells with a recombinant retroviral expression vector containing the human erbB-2 protooncogene (20) to ascertain whether gpl85crbB'2 overexpression can induce transforma tion, gp 185"hB"2 overexpressing FRTL-5 clones are not transformed, but have lost their dependence from TSH for cell growth and escaped from TGFß-induced growth arrest. MATERIALS AND METHODS Cell Cultures and Infection. The FRTL-5 (18) rat thyroid cells, that are derived from 3-week-old normal Fischer rats, were cultured in Coon's modi fied Ham's F-12 medium (W/O) supplemented with 5% heat-inactivated My- coplasma-ffee calf serum and a six-hormone mixture containing TSH, 1X 10~'°M; insulin, 10 jig/ml; hydrocortisone, 1 X 10~8 M; human transferrin, 5 mg/ml; somatostatin. 10 ng/ml: and glycyl-i-histidyl-i.-lysine acetate. 10 ng/ ml. Porcine TGFß,isoform was purchased from R & D System (Minneapolis, Mn). The PN12 viral stock used for infection of FRTL-5 cells contains an internal SV4()-carly region promoter that drives the expression of the hygro- mycin-resistance gene and a mouse MT-1 promoter that controls the expression of human c-erbB-2 complementary DNA (20). After hygromycin selection (200 /ng/ml for 4 weeks), individual clones were selected and expanded into cell lines. FRTL-5I represents one of the FRTL-5 clones infected with the control virus carrying only the hygromycin resistance gene (PN02) (20). All cell lines were grown in the continuous presence of 1 ¿¿M CdCl2 to maximally induce the expression of the c-erfoB-2 gene through the MT-1 promoter. This CdCli concentration does not interfere with the normal growth of control FRTL-5 cells (both control cells and cells growing in presence of CdCl2, 1 ^.M, present a doubling time of 32 h). Soft agar assays were performed according to the technique described (21). Iodide uptake assays were performed as described (15). Western Blot Analysis. Protein lysates (50 fig of total protein/lane) were separated by SDS-PAGE, transferred to nitrocellulose, and reacted with rabbit polyclonal anti-gpl85crhli'2 antiserum. For immunoprecipitation experiments, cells were lysed in a buffer containing 1% Triton X-100, 50 IÕIM 4-(2-hydroxy- ethyl)-l-piperazine cthanesulfonic acid. pH 7.5, 150 ITIMsodium chloride, 1.5 iTiMethyleneglycol bis (ß-aminoethyl ether)-W,(V,iV',/v"-tetraacetic acid, phos- phatase inhibitors (10 mw sodium pyrophosphate and 400 JIM sodium ortho- vanadate) and protease inhibitors (1 mw phenylmethylsulfonyl fluoride and 50 5548 Research. on January 15, 2016. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from