Effect Of Wild-Type P53 Gene On Centrosome In Leukamia K562 Cells

Recently, lots of studies have demonstrated that the initiation and progression of solid tumors are associated with the formation of abnormal centrosome, which contributes to abnormal mitosis, aneuploid and genomic instability. Abnormality of centrosome includes the abnormality of its number and structure. Numerical abnormality is associated with its increased number, over-duplication, cytocinesis failure and early departure, while the structural abnormality was due to the over-expression of centrosomal proteins or existence of abnormal phosphorylation. Further studies have indicated that p53 gene mutation and deletion, one of the major reasons for development of aneuploid and genomic instability, can lead to the upregulation of CDK (cyclin-dependent kinase, CDK) activity, deregulation of the G1 and G2 checkpoint, as well as multiple duplication and multicentric mitosis of centrosome. In recent years, the life span of the CML patients have been significantly increasing due to the clinical applications of allogeneic hematopoietic stem-cell transplantation, interferon administration and the combined chemo-therapeutics, however, there are still about 70% patients who progress into the blast crisis phase within 5 years,and the corresponding mechanism still remains unclear up to now. It is observed that besides the Philadelphia chromosome, more than 80% patients have genetical and chromosomal abnormalities such as the triploidy of 8, 19 chromosomes, while at the molecular level, p53 gene mutation occurs, whose mechanism is still unclear nowadays. What's more, studies have shown that there are abnormal centrosomes in CML patients at different stages, including numerical abnormality and structural abnormality, and the degree of abnormality which is associated with the clinical stages, is more severe in the blast crisis stage. Considering these, this study uses p53 gene as the intervention point , and takes the K562 cells with p53 gene mutation as research model to observe the alteration of centrosome number and the expression of centrosomalγ-tubulin protein, as well as the proliferation of K562 cells after the restoration of P53 protein expression by transferring wild-type P53 gene into the K562 cells, and to explore the pathogenesis of blast crisis and the possibility of application of wild-type P53 gene therapy in the treatment of chronic myeloid leukemia as well. The research was carried out in three steps as follows:1. Construction of K562 cell model expressing exogenous wild type p53 gene.The recombinant adenoviruses carrying wild-type p53 gene, mutational P53 gene and the green fluorescent protein(GFP) gene were all repeatedly amplified in 293 cells and were co-infected into K562 cells with cation polybrene after the detection of the virus titers. The infection dose of polybrene was screened. The optimal infection titer and infection time of the recombinant adenoviruses were determined by MTT assay. After the infection of K562 cells by the optimal adenovirus infection titer, mRNA and protein expression of p53 gene were determined by RT-PCR and Western blot respectively. The recombinant adenoviruses were successfully amplified repeatedly and the virus titers were 1.0×1010efu/ml,1.0×1011efu/ml and 1.0×1011efu/ml respectively. 4ug/ml polybrene was chosen. The optimal adenovirus infection titer was 20000MOI and the optimal infection time was 72 hours. p53 mRNA expression was observed at 24h after the infection of Ad5mtp53 and continued for 72 hours. What's more, P53 protein sustained to express after 72 hours of infection.2. Effect of the recombinant adenovirus-mediated wild-type p53 gene infection on the number and proteins of centrosome in K562 cells.After confirmation of successful infection by p53, the centrosomal structural proteinsγ-tubulin protein and the spindleα-tubulin protein were marked simultaneously by the indirect immunofluorescence staining, and at the same time, the expression of the centrosomalγ-tubulin protein, the mitosis and the number of centrosome were all observed under the laser confocal microscopy. Both the expression of the centrosomalγ-tubulin protein and the number of centrosomes were decreased 72 hours after infection.3. Effect of the recombinant adenovirus-mediated wild-type P53 gene infection on the proliferation of K562 cellsThe cell cycle and DNA contents of K562 cells were analyzed by flow cytometry, and the protein expressions of P21,NPM and phospho-NPM(Thr199) were detected by Western blot after the infection of K562 cells by the recombinant adenovirus-mediated wild-type P53 gene. The blockage of the cell cycle at the G0/G1 phase and the decrease of DNA content were observed after 48 and 72 hours ,and at the same time, increased P21 protein expression and decreased phospho-NPM(Thr199) protein expression were also observed ,while NPM protein didn't change after the infection of K562 cells by Ad5wtp53; however, blockage of cell cycle at the G0/G1 phase or significant change of the DNA content were not observed 48 and 72hours after the infection of K562 cells by Ad5mtp53 and Ad5GFP, and the protein expressions of P21,NPM and phospho-NPM(Thr199) didn't change either.Conclusions: There is sustained expression of P53 protein in K562 cells after its infection by recombinant adenovirus-mediated wild-type P53 gene; wide-type P53 protein can lead to the deregulation the number of centrosomes and the expression of centrosomalγ-tubulin protein in K562 cells, and it may contribute to the up-regulation of P21 protein expression, the blockage of cell cycle in G0/G1 phase, the inhibition of DNA synthesis at S phase, as well as the down-regulation of Tr199-NPM phosphorylation, all of which inhibits the excessive duplication of centrosome.