Mechanisms of SAG Inhibition of Carcinogenesis & Apoptosis

描述（由申请人提供）：抗氧化剂先前已显示可抑制由肿瘤引发剂/促进剂、DMBA/TPA或UV照射诱导的皮肤致癌作用，然而，蛋白质泛素化和降解在多阶段致癌作用中的作用在很大程度上是未知的。我们的长期目标是通过诱导一种关键分子来实现皮肤癌发生的化学预防，该分子的表达抑制了这一过程。这个关键分子，SAG（对凋亡敏感的基因）或Rbx 2/ROC 2，是一种富含半胱氨酸的蛋白质和SCF（Skp 1，Cullins，F-box蛋白）的RING组分，可能是OCX（DDB 1/Cul 4A/X-box）E3泛素连接酶。我们克隆了SAG，发现SAG是一种氧化还原诱导的抗氧化剂和E3泛素连接酶。当SAG过表达时，在体外和体内均抑制氧化还原和缺氧诱导的细胞凋亡。本申请的目的是使用JB 6表皮细胞培养体外模型和K14驱动的SAG转基因小鼠体内模型来定义SAG在皮肤致癌中的抑制作用并阐明其作用机制。中心假设是肿瘤促进剂TPA或致癌物UV诱导AP-1，而UV诱导p53。AP-1和p53都通过直接结合SAG启动子中各自的共有元件来反式激活SAG表达。在诱导后，SAG清除ROS或与SCF/DCX E3泛素连接酶的其他组分的复合物，以泛素化和降解c-Jun和细胞周期蛋白D1，从而保护表皮细胞免受DMBA/TPA或UV诱导的致癌作用。验证这一假设的具体方法是：1）阐明TPA和UV通过AP-1和p53的转录激活诱导SAG的机制; 2）确定SAG在TPA诱导的肿瘤促进和UV诱导的JB 6表皮细胞凋亡中的抑制作用; 3）阐明SAG作为抗氧化剂和E3泛素连接酶的作用机制; 4）用SAG转基因小鼠测定SAG表达抑制DMBA/TPA或UV诱导的体内皮肤癌变的程度。通过这项研究，我们将证明SAG是一种新的皮肤癌发生抑制剂，其抗氧化剂和E3连接酶活性有助于这种抑制。这里产生的癌症抗性SAG小鼠可以提供分子靶点（如AP-1）的验证，当击中时，可以防止致癌作用。此外，我们将提供一个分子基础，为未来筛选药物，可能作为化学预防剂，通过SAG诱导。

DESCRIPTION (provided by applicant): 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.