Suppression of oncogenic transformation by hypothemycin associated with accelerated cyclin D1 degradation through ubiquitin-proteasome pathway

Hypothemycin was originally isolated as an antifungal metabolite of Hypomyc es trichothecoides. Here we report that treatment on v-K-ras-transformed NI H3T3 cells (DT cells) with hypothemycin caused drastic decrease in amount o f cyclin D1 protein with concomitant prolongation of G1 phase in their cell cycle. Analysis using hypothemycin-resistant mutant of Schizosaccaromyces pombe (S. pombe) was carried out to show that S. pombe rhp6(+) (homologue o f Saccaromyces cerevisiae RAD6) and mammalian ubiquitin-conjugating enzyme 2 (ubc2) are the targets of hypothemycin or its downstream molecules in ubi quitin-conjugation process. Furthermore, in the presence of lactacystin, a specific inhibitor for proteasome, hypothemycin greatly enhanced the accumu lation of multi-ubiquitinated form of cyclin D1 in DT cells. Therefore, it is indicated that hypothemycin facilitates ubiquitinating process of cyclin DI. In terms of malignant phenotype, hypothemycin inhibited anchorage-inde pendent growth and reverted the morphology of DT cells. On the contrary, th eir morphology still remained transformed in the additional presence of lac tacystin. Our results suggest that cyclin D1 is a key molecule working down stream in ras-signaling and that the transformation can be inhibited by the compound which can activate ubiquitin-proteasome pathway including degrada tion of cyclin D1.