A fluorescent p53GFP fusion protein facilitates its detection in mammalian cells while retaining the properties of wild-type p53

Abstract

Tumor progression is often characterized by the cumulative loss of crucial cell cycle control genes and the concomitant loss of genome stability. Progressed tumors are often resistant to conventional therapies. Gene-transfer of key growth-regulatory genes, such as the p53 gene, is one potential approach to treating advanced tumors. To this end, we have produced high-titer retroviruses, based on the pCL vector system, which encode a chimeric protein consisting of human wild-type p53 and the green fluorescent protein (wtp53GFP). The fluorescent wtp53GFP protein and the wild-type p53 protein are recognized equally by several monoclonal p53-specific antibodies, have similar half-lives and function comparably in transactivating a p53-responsive element as well as in suppressing the growth of tumor cells. Additionally, due to its fluorescent nature, wtp53GFP facilitates the direct identification of cells expressing the p53 fusion protein. Combining the features of the pCL retroviral production system with the highly visible green fluorescent protein provides a potent tool for the delivery of p53 into cells and the subsequent detection of the protein, both in vitro and in vivo.