Expression of teratocarcinoma-derived growth factor-1 (TDGF-1) in testis germ cell tumors and its eects on growth and dierentiation of embryonal carcinoma cell line NTERA2/D1 Gustavo Baldassarre 1 , Annunciata Romano 2 , Franca Armenante 1,5 , Marco Rambaldi 2 , Iole Paoletti 1 , Claudia Sandomenico 3 , Stefano Pepe 3 , Stefania Staibano 4 , Gaetano Salvatore 4 , Gaetano De Rosa 4 , Maria Graziella Persico 1 and Giuseppe Viglietto 2 1 International Institute of Genetics and Biophysics, CNR, Via Marconi 12, 80125 Naples, Italy; 2 Istituto Nazionale dei Tumori di Napoli, Fondazione Senatore Pascale, Via M. Semmola, 80131 Naples, Italy; 3 Dipartimento di Endocrinologia ed Oncologia Sperimentale e Clinica and 4 Istituto di Patologia, Facolta Á di Medicina e Chirurgia, Universita Á Federico II di Napoli, Via Pansini, 80131 Naples, Italy The teratocarcinoma-derived growth factor-1 (TDGF-1) gene codes for a 188-aminoacid glycoprotein that shares structural homology with the epidermal growth factor (EGF) family of growth factors. TDGF-1 is highly expressed in the undierentiated embryonal carcinoma stem cell line NTERA2 clone D1 (NT2/D1) and its expression is downregulated in response to dierentiating agents such as retinoic acid (RA) and hexamethylen- bisacetamide (HMBA). To assess the role of TDGF-1 in the onset and/or progression of human germ cell tumors, we analysed TDGF-1 expression by Northern blot and immunostaining in a panel of 59 human germ cell tumors of dierent histological origins. We show that TDGF-1 expression is markedly elevated in a subset of human testicular germ cell tumors as compared to normal testes. TDGF-1 overexpression occurs in about 100% of tumors with non-seminomatous phenotype, such as embryonal carcinomas and malignant undierentiated teratocarci- nomas. To address the questions of how TDGF-1 (previously called CRIPTO) may aect the growth and/or the dierentiation of embryonal carcinoma cells, we have characterized the eects of exogenous recombi- nant TDGF-1 protein on the proliferation rate and dierentiation potential of NT2/D1. Exogenous TDGF-1 protein stimulated DNA synthesis and cell proliferation in both undierentiated and dierentiated NT2/D1 cells. However, TDGF-1 protein treatment was unable to block dierentiation induced by both RA and HMBA. These results suggest that TDGF-1 growth factor may represent an autocrine growth factor that may be involved in the process of development of testicular neoplasms. Keywords: TDGF-1; teratocarcinoma; seminoma; cell dierentiation Introduction Germ cell tumors (GCTs) of the testis represent a heterogeneous group of neoplasms that predominantly occur in young men. Their incidence has progressively increased in the twentieth century, since testis cancer is the most common carcinoma in the 15 ± 35 year-old age-group. Despite the advances in the managment of GCT, this disease and the accompanying treatment have a severe impact on such patients during the most productive years of their life. Histologically, GCTs comprise two main groups, seminomas and non- seminomas (Ulbright and Roth, 1994). The biological properties of these tumors make them a suitable system to study the relationship between dierentiation and transformation. However, the molecular basis of malignant transformation and expression of differen- tiated phenotypes by GCTs cells are poorly under- stood. Experimental evidence suggests that polypeptide growth factors may play a role in the growth of human tumors both in vitro and in vivo (Cross and Dexter, 1991; Salomon et al., 1995a). Growth factors have been grouped in a small number of families on the basis of their aminoacid sequence. One of these families comprises, among others, epidermal growth factor (EGF) (Carpenter and Cohen, 1990), transform- ing growth factor-a (TGF-a) (Derynck, 1988), amphir- egulin (AR) (Plowman et al., 1990) and CRIPTO-1 (Ciccodicola et al., 1989). The TDGF-1 (previously named CRIPTO) gene has been isolated from the human embryonal carcinoma cell line NT2/D1 (Ciccodicola et al., 1989). It encodes a 2.0 kb mRNA expressed in undierentiated NT2/D1 cells (Ciccodicola et al., 1989). TDGF-1 can function as a dominant transforming gene. Overexpression of TDGF-1 full-length cDNA in the NOG-8 mouse mammary epithelial cell line (Ciardiello et al., 1991b) or NIH/3T3 ®broblasts (Ciccodicola et al., 1989) results in the in vitro transformation of these cells as it confers to TDGF-1- transfected cells the ability to grow in soft agar. Complementary evidence of the causal involvement of TDGF-1 gene in tumor growth is furnished by Ciardiello and co-workers (Ciardiello et al., 1994) who have shown that TDGF-1 down-regulation by the use of antisense cDNA-expressing vector strongly reduces the anchorage-independent growth in vitro and tumor development in vivo of the colon carcinoma cell line GEO. TDGF-1 is overexpressed in 70% of human primary colorectal carcinomas, in 60% of hepatic metastasis from colon cancers (Ciardiello et al., 1991a) and in 75% of mammary carcinomas (Qi et Correspondence: MG Persico 5 Present address: Institute of Biological Chemistry, University of Verona, Strada Le Grazie, 8, 37134 Verona, Italy Received 21 March 1997; revised 7 May 1997; accepted 7 May 1997 Oncogene (1997) 15, 927 ± 936 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00