Enhanced gene transfer and cell death following p53 gene transfer using photochemical internalisation of glucosylated PEI-DNA complexes.
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.
Ndoye
Alioune
A
Laboratoire de Recherche en Oncologie, Centre Alexis Vautrin, EA3452, Vandoeuvre-les-Nancy, France.
Merlin
Jean-Louis
JL
Leroux
Agnès
A
Dolivet
Gilles
G
Erbacher
Patrick
P
Behr
Jean-Paul
JP
Berg
Kristian
K
Guillemin
François
F
D017209:Apoptosis; D053938:DNA Fragmentation; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007202:Indicators and Reagents / Q000737:chemistry; D008856:Microscopy, Fluorescence; D010777:Photochemistry; D010957:Plasmids; D011094:Polyethyleneimine / Q000031:analogs & derivatives*; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay / Q000379:methods; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D007202:Indicators and Reagents; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine