The MAGE-A, MAGE-B, and MAGE-C protein families comprise the class-I MAGE/cancer testes antigens, a group of highly homologous proteins whose expression is suppressed in all normal tissues except developing sperm. Aberrant expression of class I MAGE proteins occurs in melanomas and many other malignancies, and MAGE proteins have long been recognized as tumor-specific targets; however, their functions have largely been unknown. Here, we show that suppression of class I MAGE proteins induces apoptosis in the Hs-294T, A375, and S91 MAGE-positive melanoma cell lines and that members of all three families of MAGE class I proteins form complexes with KAP1, a scaffolding protein that is known as a corepressor of p53 expression and function. In addition to inducing apoptosis, MAGE suppression decreases KAP1 complexing with p53, increases immunoreactive and acetylated p53, and activates a p53 responsive reporter gene. Suppression of class I MAGE proteins also induces apoptosis in MAGE-Apositive, p53wt/wt parental HCT 116 colon cancer cells but not in a MAGE-A-positive HCT 116 p53 À/À variant, indicating that MAGE suppression of apoptosis requires p53. Finally, treatment with MAGE-specific small interfering RNA suppresses S91 melanoma growth in vivo, in syngenic DBA2 mice. Thus, class I MAGE protein expression may suppress apoptosis by suppressing p53 and may actively contribute to the development of malignancies and by promoting tumor survival. Because the expression of class I MAGE proteins is limited in normal tissues, inhibition of MAGE antigen expression or function represents a novel and specific treatment for melanoma and diverse malignancies. [Cancer Res 2007;67(20):9954-62]

Glycosylated natural products are reliable platforms for the development of many front-line drugs, yet our understanding of the relationship between attached sugars and biological activity is limited by the availability of convenient glycosylation methods. When a universal chemical glycosylation method that employs reducing sugars and requires no protection or activation is used, the glycorandomization of digitoxin leads to analogs that display significantly enhanced potency and tumor specificity and suggests a divergent mechanistic relationship between cardiac glycosideinduced cytotoxicity and Na ؉ ͞K ؉ -ATPase inhibition. This report highlights the remarkable advantages of glycorandomization as a powerful tool in glycobiology and drug discovery.carbohydrate ͉ natural product ͉ sugar

To construct a cyclin-luciferase fusion protein (pSP cyc-luc), the N-terminal sequence of Xenopus laevis cyclin B1, including amino acids 2-97, was amplified by PCR, digested with BstEII, and ligated into the pSP-lucNF expression vector (Promega).The resulting vector was sequence verified. The fusion protein was expressed by coupled in vitro transcription and translation in reticulocyte lysate using the SP6-TNT Coupled Reticulocyte Lysate System (Promega) and flash frozen in liquid nitrogen until the time of use. The parental pSP-lucNF vector was used to express unmodified luciferase.A vector for expression of cyclin-luciferase in E. coli (pET cyc-luc) was also constructed; this protein behaved identically in all assays to the protein expressed in reticulocyte lysate, but could be made in higher quantities necessary for screening. pSP cyc-luc was digested with HindIII and XhoI. The resulting 1949 bp fragment containing the cyclin B1-luciferase sequence was ligated into the pET 28b expression vector