[p16INK4a expression mediated by recombinant adenovirus can induce senescence of A549 cells].
OBJECTIVE
To construct E1-deletion and replication-defective human type 5 recombinant adenovirus vector and to study the effect of p16INK4a on proliferation and aging of A549 cells.


METHODS
p16INK4a cDNA was cloned into pAdCMV to construct recombinant pAdCMV p16INK4a, which was co-transfected into 293 cell together with pJM17. The recombinant p16INK4a adenovirus (Ad-p16INK4a) was generated by homologous recombination and identified with duplex PCR. Lung cancer cell A549, which has a homozygous deletion of p16INK4a gene, was infected with the prepared Ad-p16INK4a virus. X-gal staining and TRAP-ELISA were used for detecting senescence-associated beta-galactosidase and telomerase activities in A549 cells.


RESULTS
Immunohistochemical staining and Western blot indicated that p16INK4a gene was transferred into A549 cell with more than 95% efficiency by recombinant adenovirus and p16INK4a protein was expressed at a high level- p16INK4a could markedly inhibit growth of A549 cells, induced expression of senescence-associated beta-galactosidase and suppressed telomerase activity in A549 cells.


CONCLUSIONS
Recombinant adenovirus vector could efficiently mediate transfer and expression of foreign genes in human cell and could be used for gene immunization and gene therapy; p16INK4a could inhibit A549 cell growth and induce its replicative senescence.
Bai
Xue-yuan
XY
Department of Nephrology, Chinese PLA General Hospital, Beijing 100853, China. bai_xueyuan@yahoo.com
Che
Feng-xiang
FX
Chen
Xiang-mei
XM
Meng
Ling-ying
LY
Zhou
Yu
Y
D000256:Adenoviridae / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D016922:Cellular Senescence; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D011995:Recombination, Genetic; D014162:Transfection
D019941:Cyclin-Dependent Kinase Inhibitor p16