Mutations in the human Tsc1 and Tsc2 genes predispose to tuberous sclerosis complex (TSC), a disorder characterized by the wide spread of benign tumors. Tsc1 and Tsc2 proteins form a complex and serve as a GTPase-activating protein (GAP) for Rheb, a GTPase regulating a downstream kinase, mTOR. The genome of Schizosaccharomyces pombe contains tsc1 1 and tsc2 1

Rheb, a Ras-like small GTPase conserved from human to yeast, controls Tor kinase and plays a central role in the regulation of cell growth depending on extracellular conditions. Rhb1 (a fission yeast homolog of Rheb) regulates amino acid uptake as well as response to nitrogen starvation. In this study, we generated two mutants, rhb1-DA4 and rhb1-DA8, and characterized them genetically. The V17A mutation within the G1 box defined for the Ras-like GTPases was responsible for rhb1-DA4 and Q52R I76F within the switch II domain for rhb1-DA8. In fission yeast, two events-the induction of the meiosis-initiating gene mei2 1 and cell division without cell growth-are a typical response to nitrogen starvation. Under nitrogen-rich conditions, Rheb stimulates Tor kinase, which, in turn, suppresses the response to nitrogen starvation. While amino acid uptake was prevented by both rhb1-DA4 and rhb1-DA8 in a dominant fashion, the response to nitrogen starvation was prevented only by rhb1-DA4. rhb1-DA8 thereby allowed genetic dissection of the Rhebdependent signaling cascade. We postulate that the signaling cascade may branch below Rhb1 or Tor2 and regulate the amino acid uptake and response to nitrogen starvation independently.

Abstract. SC1, an immunoglobulin superfamily cell adhesion molecule, is expressed in embryonic tissues and plays an important role in development through its cell adhesive activity. SC1 is also found in a variety of tumors and its expression is associated with a poor prognosis. The expressional patterns of SC1 were examined in sporadic cases of canine mammary gland tumors and it was found that this molecule is enriched in adenocarcinomas and is weaker in benign mixed tumors. SC1 might therefore be involved in the malignancy and progression of canine mammary gland tumors. To confirm this paradigm, the mammary gland cell line JYG-B was used as the recipient of SC1 cDNA. The resulting SC1-transfected cells were subsequently analyzed using a convenient in vitro model system. The self-aggregation activity of SC1-transfected cells was significantly increased and was blocked by an anti-SC1 antibody generated by hyper-immunized ostrich yolk. In addition, cell locomotion assays revealed an enhanced migration activity of SC1-transfected cells on SC1-coated transwell chambers. The in vivo activities of the cells were examined by subcutaneous implantation into nude mice. Tumor growth was significantly promoted in the mice after implantation with SC1-transfected cells, in comparison to parental-and mocktransfectants. This growth was inhibited by oral administration of gold-ion water. The invasion of SC1-transfectants into the surrounding muscular and adipose tissues was rigorously enhanced. These findings suggest that SC1 might promote the progression of mammary gland tumor cells by increasing cell adhesion.