[Effect of wild-type p53 gene on the number and proteins of centrosome in leukemic K562 cells].
OBJECTIVE
To observe the effect of recombinant adenovirus-mediated wild-type p53 gene on the number and proteins of centrosome in K562 cells. To explore the possibility of application of wild-type p53 gene therapy in the treatment of chronic myeloid leukemia.


METHODS
The recombinant adenoviruses carrying wild-type p53 gene (Ad5 wtp53), mutant p53 gene (Ad5 mtp53) or the green fluorescent protein (GFP) gene was repeatedly amplified and co-infected into K562 cells with cation polybrene. The optimal infection titer and infection time of the recombinant adenoviruses were determined by MTT assay, p53 mRNA and protein expression were determined by RT-PCR and Western blot respectively. The centrosomal structural protein gamma-tubulin and the spindle protein alpha-tubulin were marked simultaneously by indirect immunofluorescence staining, and the expression of the centrosomal gamma-tubulin protein, the mitosis and the number of centrosome were observed under the laser confocal microscopy.


RESULTS
Infection efficiency with recombinant adenoviruses was facilitated by polybrene in K562 cells, and 4 microg/ml polybrene was chosen. The optimal adenovirus infection titer was 20,000 MOI and the optimal infection time was 72 hours. p53 mRNA and P53 protein can be expressed in K562 cells by Ad5wtp53 and Ad5mtp53. Both the expression of the centrosomal gamma-tubulin protein and the number of centrosomes were decreased after Ad5wtp53 infection.


CONCLUSIONS
There is sustained expression of P53 protein in K562 cells after its infection by Ad5wtp53. Wild-type P53 protein can lead to the down-regulation of the number of centrosomes and the expression of centrosomal gamma-tubulin protein in K562 cells.
Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, Chongqing Medical University, Chongqing 400016, China.
Hong-bin
HB
Wang
Wen-li
WL
Feng
Shi-feng
SF
Huang
Wen-jun
WJ
Tian
Wei-xi
WX
Cao
Zong-gan
ZG
Huang
D000256:Adenoviridae / Q000235:genetics; D018385:Centrosome / Q000378:metabolism*; D016158:Genes, p53 / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D014404:Tubulin; D016159:Tumor Suppressor Protein p53