ABSTRACT
BACKGROUND: The use of integrating viral vectors in Gene therapy clinical trials has pointed out the problem of the deleterous effect of the integration of the ectopic gene to the cellular genome and the safety of this strategy. We proposed here a way to induce the death of gene modified cells upon request by acting on a pro-apoptotic protein cellular localization and on the activation of its apoptotic function. RESULTS: We constructed an adenoviral vector coding a chimeric p53 protein by fusing p53 sequence with the 21 COOH term amino acids sequence of H-Ras. Indeed, the translation products of Ras genes are cytosolic proteins that become secondarily associated with membranes through a series of post-translational modifications initiated by a CAAX motif present at the C terminus of Ras proteins. The chimeric p53HRCaax protein was farnesylated efficiently in transduced human osteosarcoma p53-/- cell line. The farnesylated form of p53 resided mainly in the cytosol, where it is non-functional. Farnesyl transferase inhibitors (FTIs) specifically inhibited farnesyl isoprenoid lipid modification of proteins. Following treatment of the cells with an FTI, p53HRCaax underwent translocation into the nucleus where it retained transcription factor activity. Shifting p53 into the nucleus resulted in the induction of p21waf1/CIP1 and Bax transcription, cell growth arrest, caspase activation and apoptosis. CONCLUSION: Artificial protein farnesylation impaired the transcriptional activity of p53. This could be prevented by Farnesyl transferase inhibition. These data highlight the fact that the artificial prenylation of proteins provides a novel system for controlling the function of a transactivating factor.
Subject(s)
Protein Prenylation , Recombinant Fusion Proteins/genetics , Transcriptional Activation , Tumor Suppressor Protein p53/antagonists & inhibitors , Adenoviridae/genetics , Apoptosis , Cell Line, Tumor , Cell Membrane/chemistry , Cell Proliferation , Enzyme Inhibitors/pharmacology , Farnesyltranstransferase/antagonists & inhibitors , Genetic Vectors , Humans , Mutation , Protein Prenylation/drug effects , Protein Structure, Tertiary , Proto-Oncogene Proteins p21(ras)/genetics , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/metabolism , Transcriptional Activation/drug effectsABSTRACT
Shope fibroma virus (SFV) is one of the few poxviruses that induce cutaneous tumours, whereas myxoma virus, a closely related leporipoxvirus, does not. However, both have a virally encoded homologue of the epidermal growth factor (namely SFGF and MGF, respectively) that is considered to be crucial for poxvirus tumorigenesis. In this study, the role of viral growth factors in the context of infection with SFV, a tumorigenic leporipoxvirus, was investigated. An SFV mutant was engineered with the sfgf gene deleted and replaced with mgf. Macroscopic, histological and cytological examinations led to the conclusion that growth factors are indeed important for the development and maintenance of fibromas, provided that they are expressed in the proper viral context. However, they are not exchangeable and MGF cannot substitute for SFGF in the genesis of fibromas. It is likely that factors other than viral epidermal growth factor homologues influence the development of tumours.
