¹Laboratorio di Biochimica e Biologia Molecolare - Stazione Zoologica "Anton Dohrn" Villa Comunale, 80121 Napoli, Italy

²CRIBI Biotechnology Center, Dipartimento di Biologia, Universita' degli Studi di Padova, viale G. Colombo 3, Padua, 35121, Italy

Correspondence to: R Di Lauro, Laboratorio di Biochimica e Biologia Molecolare - Stazione Zoologica "Anton Dohrn" Villa Comunale, 80121 Napoli, Italy

Expression of oncogenic v-H-Ras in the thyroid cell line FRTL-5 (FRTL-5^Ras) results in uncontrolled proliferation, loss of thyroid-specific gene expression and tumorigenicity. Concomitant expression of constitutively activated MEK and Rac, two major H-Ras downstream effectors, in FRTL-5 (FRTL-5^MEK/Rac) recapitulates H-Ras effects on proliferation and morphology. In contrast to FRTL-5^Ras, however, FRTL-5^MEK/Rac cells remain differentiated and are not tumorigenic. To find H-Ras induced genes potentially responsible for tumorigenicity and loss of differentiation, we have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, between de-differentiated and tumorigenic FRTL-5^Ras cells and differentiated and non-tumorigenic FRTL-5^MEK/Rac cells. We examined 800 of the cDNA clones obtained after subtraction and verified their levels of expression in the two cell lines by reverse northern, identifying 337 H-Ras induced genes. By sequence analysis, we clustered 57 different genes. Among these, 39 were known genes (involved in diverse signal transduction processes regulating mitogenic activity, cell survival, cytoskeletal reorganization, stress response and invasion) while the remaining 18 clones were novel genes. Among the 57 H-Ras specific clones, we identified those genes whose expression is induced early by H-Ras. We suggest that these immediate-early genes may play a crucial role in H-Ras-mediated transformation in thyroid epithelial cells. Oncogene (2001) 20, 2281-2290.

thyroid; tumorigenicity; differentiation; subtraction library; adenovirus