Cyclin D1 regulates G1 cell cycle progression and is often aberrantly expressed in carcinogenesis. Our prior work highlighted cyclin D1 as a molecular pharmacologic target for cancer therapy and chemoprevention. We previously reported degradation of cyclin D1 as a chemopreventive mechanism conferred by retinoid treatment and by activation of the ubiquitin-activating enzyme E1-like protein (UBE1L). The subsequent induced G1 arrest was hypothesized to permit repair of genomic DNA damage caused by carcinogens. To better understand this regulation of cyclin D1, those residues responsible for conferring ubiquitin-dependent degradation of cyclin D1 were investigated. Eighteen lysine residues present in cyclin D1 were engineered with individual or multiple mutations (arginine transversions) and these species were transfected into BEAS-2B human bronchial epithelial (HBE) cells to evaluate stabilities following all-trans-retinoic acid (RA) treatments. Specific single or double lysine mutations stabilized cyclin D1 to an extent. Yet, substitution of at least 9 lysine residues of cyclin D1 protein surrounding the cyclin box domain were required to cause a prominent inhibition of RA-mediated degradation. Mutation of the additional lysines flanking this region conferred even greater stabilization of cyclin D1. As expected, mutation of all cyclin D1 lysines blocked polyubiquitination. Interestingly, these lysine residues also played an important role in UBE1L-mediated cyclin D1 degradation. In addition, ubiquitination-resistant mutants preferentially localized cyclin D1 to the nucleus, directly implicating subcellular localization in the regulation of cyclin D1 degradation. Taken together, these findings highlight a molecular pharmacological basis for triggering cyclin D1 degradation. These observations have implications for targeting cyclin D1 in cancer therapy and chemoprevention.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA