Reduced transduction efficiency of adenoviral vectors expressing human p53 gene by repeated transduction into glioma cells in vitro.
OBJECTIVE
Recombinant adenoviral vectors are widely used in clinical and experimental studies to treat malignant tumors. Recently, host immune responses have been proposed as a major limitation in using adenoviral vectors for repeated gene delivery. We demonstrate another limitation unrelated to host immunity.


METHODS
We repeatedly transduced an adenoviral vector expressing the human p53 gene (AxCIhp53) into U373MG, a p53-susceptible cell line, and established the AxCIhp53-resistant cell line U373R. Most U373R cells survived even after AxCIhp53 treatment due to reduced transduction efficiency. Expression levels of adenovirus receptors were estimated to investigate the cause of reduced transduction efficiency. The mutant vector was used to overcome the resistance.


RESULTS
The transduction efficiency of an adenoviral vector possessing the reporter LacZ gene (AxCAZ2-F/wt) for U373R cells was 25.4-fold less than that for parent cells. The expression levels of integrins alpha(v)beta(3) and alpha(v)beta(5) were found to be decreased in U373R cells without affecting the expression levels of Coxsackievirus and adenovirus receptor. The mutant vector AxCAZ2-F/K20, with a linker and a stretch of 20 lysine residues at the COOH-terminal of the fiber protein, improved the transduction efficiency of U373R cells to 12.6-fold of that of AxCAZ2-F/wt. A mutant vector carrying the p53 gene, AxCAhp53-F/K20, dramatically induced apoptosis in U373R cells.


CONCLUSIONS
Glioma cells expressing low levels of adenovirus receptors might survive and proliferate to recur after repeated adenoviral transduction, even if the adenoviral transduction is effective at first. Changing the tropism of vectors is a potent method to overcome resistance.
Yamamoto
Shinji
S
Department of Molecular Biotherapy Research, Cancer Chemotherapy Center, Cancer Institute, Tokyo 170-8455, Japan. yamamoto.nsrg@med.tmd.ac.jp
Yoshida
Yoko
Y
Aoyagi
Masaru
M
Ohno
Kikuo
K
Hirakawa
Kimiyoshi
K
Hamada
Hirofumi
H
D000256:Adenoviridae / Q000235:genetics*; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000628:therapy; D002448:Cell Adhesion; D002455:Cell Division; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D017931:DNA Primers / Q000737:chemistry; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D016158:Genes, p53 / Q000502:physiology*; D005822:Genetic Vectors / Q000502:physiology*; D005910:Glioma / Q000235:genetics* / Q000378:metabolism / Q000628:therapy; D006801:Humans; D016023:Integrins / Q000235:genetics*; D009154:Mutation; D012333:RNA, Messenger / Q000378:metabolism; D011991:Receptors, Virus / Q000235:genetics*; D019030:Receptors, Vitronectin; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D017931:DNA Primers; D016023:Integrins; D012333:RNA, Messenger; D011991:Receptors, Virus; D019030:Receptors, Vitronectin; C064660:integrin alphaVbeta5