Antioxidants have been previously shown to inhibit skin carcinogenesis induced by tumor initiator/promoter, DMBA/TPA, or UV irradiation, the role of protein ubiquitination and degradation in multistage carcinogenesis is, however, largely unknown. Our long-range goal is to achieve chemo-prevention of skin carcinogenesis through induction of a critical molecule whose expression inhibits such a process. This critical molecule, SAG (Sensitive to Apoptosis Gene) or Rbx2/ROC2, is a cysteine-rich protein and a RING component of SCF (Skp1, Cullins, F-box proteins), possibly of OCX (DDB1/Cul4A/X-box) E3 ubiquitin ligases. We cloned SAG and found that SAG is a redox inducible antioxidant and an E3 ubiquitin ligase. When over-expressed, SAG inhibits apoptosis induced by redox and hypoxia both in vitro and in vivo. The objective of this application is to define an inhibitory role of SAG in skin carcinogenesis and to elucidate its mechanism of action, using a JB6 epidermal cell culture in vitro model and a K14 driven SAG transgenic mouse in vivo model. The central hypothesis is that tumor promoter TPA or carcinogen UV induces AP-1, whereas UV induces p53. Both AP-1 and p53 transactivate SAG expression through a direct binding to their respective consensus elements in the SAG promoter. Upon induction, SAG scavenges ROS or complexes with other components of SCF/DCX E3 ubiquitin ligases to ubiquitinate and degrade c-Jun and cyclin D1, thus protecting epidermal cells from DMBA/TPA- or UV-induced carcinogenesis. The specific a/msto test the hypothesis are 1) to elucidate the mechanism of SAG induction by TPA and UV through transcriptional activation by AP-1 and p53; 2) to define an inhibitory role of SAG in TPA-induced tumor promotion and in UV-induced apoptosis in JB6 epidermal cells; 3) to elucidate mechanism of SAG action as an antioxidant and an E3 ubiquitin ligase; 4) to use SAG transgenic mice to determine the extent to which SAG expression inhibits in vivo skin carcinogenesis induced by DMBA/TPA or UV. Through this research, we will demonstrate that SAG is a novel inhibitor of skin carcinogenesis and that both its antioxidant and E3 ligase activities contribute to such an inhibition. The cancer resistant SAG mice generated here can provide validation of molecular targets (such as AP-1) that when hit, function to prevent carcinogenesis. Furthermore, we will provide a molecular basis for future screening of drugs that may act as chemo-prevention agents via SAG induction. ? ? ?
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