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 Hypomyces trichothecoides. Here we report that treatment on v-K-ras-transformed NIH3T3 cells (DT cells) with hypothemycin caused drastic decrease in amount of cyclin D1 protein with concomitant prolongation of G1 phase in their cell cycle. Analysis using hypothemycin-resistant mutant of Schizosaccharomyces pombe (S. pombe) was carried out to show that S. pombe rhp6+ (homologue of Saccharomyces cerevisiae RAD6) and mammalian ubiquitin-conjugating enzyme 2 (ubc2) are the targets of hypothemycin or its downstream molecules in ubiquitin-conjugation process. Furthermore, in the presence of lactacystin, a specific inhibitor for proteasome, hypothemycin greatly enhanced the accumulation of multi-ubiquitinated form of cyclin D1 in DT cells. Therefore, it is indicated that hypothemycin facilitates ubiquitinating process of cyclin D1. In terms of malignant phenotype, hypothemycin inhibited anchorage-independent growth and reverted the morphology of DT cells. On the contrary, their morphology still remained transformed in the additional presence of lactacystin. Our results suggest that cyclin D1 is a key molecule working downstream in ras-signaling and that the transformation can be inhibited by the compound which can activate ubiquitin-proteasome pathway including degradation of cyclin D1.