SUPPRESSION OF TUMORIGENICITY BY THE WILD-TYPE TUBEROUS-SCLEROSIS-2 (TSC2) GENE AND ITS C-TERMINAL REGION

The Tsc2 gene, which is mutationally inactivated in the germ line of s ome families with tuberous sclerosis, encodes a large, membrane-associ ated GTPase activating protein (GAP) designated tuberin. Studies of th e the Eker rat model of hereditary cancer strongly support the role of Tsc2 as a tumor suppressor gene. In this study, the biological activi ty of tuberin was assessed by expressing the wild-type Tsc2 gene in tu mor cell lines lacking functional tuberin and also in rat fibroblasts with normal levels of endogenous tuberin. The colony forming efficienc y of Eker fat-derived renal carcinoma cells was significantly reduced following reintroduction of wild-type Tsc2. Tumor cells expressing the transfected Tsc2 gene became more anchorage-dependent and lost their ability to form tumors in severe combined immunodeficient mice. At the cellular level, restoration of tuberin expression caused morphologica l changes characterized by enlargement of the cells and increased cont act inhibition, As with the full-length Tsc2 gene, a clone encoding on ly the C terminus of tuberin (amino acids 1049-1809, including the GAP domain) was capable of reducing both colony formation and in vivo tum origenicity when transfected into the Eker rat tumor cells, In normal Rat1 fibroblasts, conditional over-expression of tuberin also suppress ed colony formation and cell growth in vitro, These results provide di rect experimental evidence for the tumor suppressor function of Tsc2 a nd suggest that the tuberin C terminus plays an important role in this activity.