Antiproliferation Effect of Beta-TrCP Ubiquitin Ligase Mediated BCR-ABL Protein Degradation in Leukemia K562 Cells / 中国肿瘤临床

ABSTRACT

Objective: The BCR-ABL fusion gene induced by reciprocal translocation of t (9; 22) (q34; q11) plays an important role in the pathogenesis of chronic myeloid leukemia (CML). Using recombinant ade-noviruses carrying the N-terminal oligomerizaton domain (OD) of the BCR/ABL and chimeric ubiquitin ligase β-TrCP, this study was to investigate the effect of the targeted degradation of oncoprotein BCR-ABL by Ubiqui- tin-Proteasome System on the proliferation of leukemia call line K562. Methods: The recombinant adenovirus-es carrying wild-type β-TrCP gene (Ad5β-TrCP-OD-HA), mutational β-TrCP gene (Ad5 A F-TrCP-OD-HA)and green fluorescent protein gene (Ad5GFP)were amplified in 293 calls and co-infected into K562 cells respec- tively. The rates of infection were analyzed by flow cytometry (FCM). Recombinant protein and BCR-ABL ex-pression was detected by Western blot. Cell proliferation was determined by cell counting and methylcellu- cose clonal cell culture. Cell cycle was observed through FCM. Untreated K562 cells were used as blank con-trols. Result: The leukemia K562 cell lines with exogenous recombinant β-TrCP-OD-HA and F-TrCP-OD-HA gene were established. The infection rates in the three groups were over 66.4% and recombinant protein sus-tained to be expressed. Ad5β-TrCP-OD-HA down-regulated the expression of BCR-ABL and inhibited prolifer-ation of K562 cells. FCM showed that the percentage of cells at S phase was decreased to 10.88%±2.42%, while that of cells at G_0/G_1 was increased to 85.6%±5.61%, with a significant difference (P<0.05). No changes were found in the cell cycle in groups of Ad5 △ F-TrCP-OD-HA and Ad5GFP. Conclusion: There is sustained ex-pression of recombinant β-TrCP-OD-HA protein in K562 cells infected by recombinant adenovirus.β-TrCP-OD-HA could inhibit the proliferation and clonogenicity of K562 cells through targeted degradation of oncoprotein BCR-ABL and arresting the progression of call cycle.