Adenoviral p53 gene therapy promotes heat-induced apoptosis in a nasopharyngeal carcinoma cell line.
BACKGROUND
It has previously been demonstrated that Ad5CMV-p53 gene transfer, either used alone or delivered concomitantly with ionizing radiation, resulted in cytotoxicity mediated by apoptosis in nasopharyngeal carcinoma (NPC) cell lines. In this study, a novel approach was evaluated of combining Ad5CMV-p53 gene therapy with hyperthermia (HT), in the CNE-1 NPC cell line, which harbours a mutation in codon 249 of the p53 gene.


METHODS
CNE-1 cells were infected using either Ad5CMV-p53 or Ad5CMV-B-gal, followed, 24 h later, by HT (43 degrees C x 0-2 h). Protein was extracted for Western blot analysis, and apoptosis was evaluated using acridine-orange ethidium bromide staining, followed immediately by fluorescent microscopy examination for the proportion of cells displaying morphologic features of apoptosis.


RESULTS
Ad5CMV-p53 gene therapy combined with HT resulted in a dose-dependent cytotoxicity with less than 1% clonogenic survival when 10 pfu/cell of Ad5CMV-p53 was combined with 2 h heating at 43 degrees C. Western blotting demonstrated that treatment with Ad5CMV-p53 resulted in the rapid expression of p53, which was minimally affected by HT. The inducible form of hsp70 was maximally expressed at 48 h post-HT, with minimal effect when cells were additionally treated with Ad5CMV-p53. Clonogenic cytotoxicity was associated with the development of apoptosis, with up to 70% of CNE-1 cells displaying morphologic features of apoptosis after the combination treatments.


CONCLUSIONS
Based on the shapes of the clonogenic survival curves, Ad5CMV-p53 gene therapy and HT appear to interact in an additive manner, suggesting the therapeutic potential of this combined treatment approach for patients with NPC.
Qi
V
V
Department of Research, Princess Margaret Hospital/Ontario Cancer Institute, University Health Network, Toronto, Canada.
Weinrib
L
L
Ma
N
N
Li
J H
JH
Klamut
H
H
Liu
F F
FF
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects*; D015153:Blotting, Western; D002470:Cell Survival / Q000528:radiation effects; D002999:Clone Cells / Q000473:pathology / Q000528:radiation effects; D015972:Gene Expression Regulation, Neoplastic; D018014:Gene Transfer Techniques; D016158:Genes, p53 / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006979:Hyperthermia, Induced; D009303:Nasopharyngeal Neoplasms / Q000235:genetics* / Q000473:pathology* / Q000628:therapy; D009363:Neoplasm Proteins / Q000032:analysis / Q000378:metabolism; D013696:Temperature; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D009363:Neoplasm Proteins; D016159:Tumor Suppressor Protein p53