Abstract
Background
p53 is frequently mutated in many cancers including human head and neck squamous cell carcinoma and pancreatic cancer. In tumor models, wild‐type (wt) p53 gene transfer induces apoptosis and tumor regression in vivo, justifying the extensive clinical investigation of p53 gene therapy.
Methods
p53 nonviral‐mediated gene transfer was achieved using glucosylated polyethylenimine (PEI) in conjunction with photochemical internalisation (PCI). Experimental conditions were optimised using the green fluorescent protein (GFP) as a reporter. p53 gene transfer was then evaluated using semi‐quantitative RT‐PCR in p53‐deleted PANC3 and p53‐mutated FaDu cell lines. Following gene transfer, induction of apoptosis was investigated using phosphatidylserine externalisation and nuclear fragmentation assays. Induction of long‐term cell death was analysed using colony‐forming assays.
Results
PCI was found to enhance GFP gene transfer after 48 h in both cell lines. Whether using glucosylated‐PEI alone or associated with PCI, p53 gene transfer was achieved with subsequent recovery of p53 mRNA expression in PANC3 cells and a significant 4‐fold increase in p53 mRNA expression in FaDu cells. PCI was found to further enhance p53 mRNA expression by 2.3‐fold in PANC3 cells. Spontaneous induction of apoptosis following wt‐p53 gene transfer was achieved in both cell lines. PCI was found to enhance apoptosis up to levels similar to those achieved with chemotherapy. As a consequence, long‐term cell death was significantly enhanced after wt‐p53 gene transfer when PCI was used in both cell lines, yielding up to 60% cell death.
Conclusions
PCI increases glucosylated‐PEI‐mediated p53 gene transfer, apoptosis as well as cell death in mutant p53 human cancer cells. Copyright © 2004 John Wiley & Sons, Ltd.