Appella, Ettore

Abstract A p53-dependent mouse spindle checkpoint has been reported since 1995. However the underlying mechanism is still unknown. Here, we found that p53 formed complexes with CHAMP and MAD2L2. Immunofluorescence analysis revealed that p53, CHAMP and MAD2L2 colocalized at spindle fibres from pro-metaphase to anaphase. CHAMP binds MAD2L2 and antagonizes its inhibition function on APC/C. We discovered that p53 enhanced the inhibition of MAD2L2 by CHAMP. P53 knock-down decreased M-phase index. Exogenous expression of p53 increased M-phase index and extended remarkable length of time in M phase. Notably, the regulation of p53 on M phase index was completely abrogated by either CHAMP or MAD2l2 knock-down, suggesting that the regulation of p53 on spindle assembly checkpoint pathway is dependent on CHAMP and MAD2L2. These results demonstrate that p53 participates in a mitotic checkpoint that ensures the maintenance of genetic fidelity during development. Citation Format: Piyan Zhang, Erica Ungewitter, Ettore Appella, Heidi Scrable. Tumor suppressor p53 is involved in a CHAMP-dependent spindle assembly checkpoint pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-121. doi:10.1158/1538-7445.AM2013-LB-121

Abstract The majority of CTL epitopes are derived from intracellular proteins that are degraded in the cytoplasm by proteasomes into peptides that are transported into the endoplasmic reticulum by the TAP complex. These peptides can be further processed into the optimal size (8–10 residues) for binding with nascent MHC class I molecules, generating complexes that are exported to the cell surface. Proteins or peptides containing CTL epitopes can be introduced into the cytoplasm of APCs by linking them to membrane-translocating Trojan carriers allowing their incorporation into the MHC class I Ag-processing pathway. The present findings suggest that these “Trojan” Ags can be transported into the endoplasmic reticulum in a TAP-independent way where they are processed and trimmed into CTL epitopes. Furthermore, processing of Trojan Ags can also occur in the trans-Golgi compartment, with the participation of the endopeptidase furin and possibly with the additional participation of a carboxypeptidase. We believe that these findings will be of value for the design of CTL-inducing vaccines for the treatment or prevention of infectious and malignant diseases.

Abstract We describe in this study a strategy to produce synthetic vaccines based on a single polypeptide capable of eliciting strong immune responses to a combination CTL and Th epitopes with the purpose of treating malignancies or preventing infectious diseases. This strategy is based on the capacity of Trojan Ags to deliver exogenous Ags into the intracellular compartments, where processing into MHC-binding peptides takes place. Our previous work demonstrated that Trojan Ags containing a CTL epitope localized to intracellular compartments, where MHC class I-binding peptides were generated in a TAP-independent fashion by the action of various exopeptidases and the endopeptidase furin. In this study, we report that Trojan Ags containing several CTL epitopes joined via furin-sensitive linkers generated all of the corresponding MHC class I-binding peptides, which were recognized by CTL. However, Trojan Ags prepared with furin-resistant linkers failed to produce the MHC class I-binding peptides. We also present data indicating that Trojan Ags bearing both CTL and Th epitopes can generate the corresponding MHC class I- and II-binding peptides, which are capable of stimulating T cell responses. Most significantly, in vivo vaccination of mice with a single injection of multiepitope Trojan Ags resulted in strong CTL and Th responses that translated into significant antitumor responses in a model of malignant melanoma. The overall results indicate that Trojan Ags prepared with furin-sensitive linkers are ideal candidates for producing synthetic multiepitope vaccines for the induction of CTL and Th responses that could be used against a variety of diseases, including cancer.