Effects Of FABD And Coiled-Coil Domain On Bcr/Abl Cytoplasm Localization And Its Function

Objective The pathology of chronic myeloid leukemia(CML)is the formation of Bcr/Abl fusion protein which localizes mainly in cytoplasm.Bcr/Abl protein can activate various pathways such as Ras,PI3 K,and Stat5,promote CML cells proliferation and inhibit cell apoptosis.In previous studies,we designed a novel drug delivery system RATS to induce Bcr/Abl into the nuclear and promote apoptosis through P73 pathway.The fact that Bcr/Abl localizes in cytoplasm resulting in malignant transformation has shown the significance of Bcr/Abl localization in CML development and treatment,however the underlying mechanism remains unknown.F-actin binding domain and Coiled-coil domain are two important domains in Bcr/Abl.F-actin binding domain binds to F-actin directly in cytoplasm.The deletion of FABD fails to induce Bcr/Abl into nuclear,instead,it only changes its distribution.Coiled-coil domain mediates the tetramers formation of Bcr/Abl.The 63 amino acids of N-terminal fuses to Abl and leads to entrapment in cytoplasm.It has been concluded that CC domain plays an important role in Bcr/Abl cytoplasm localization.In this paper,weconstructed a FABD deletion and coiled-coil domain deletion mutant adenovirus.By using 293 T as cell model,we mainly explored the effect of F-actin binding domain and coiled-coil domain on Bcr/Abl cytoplasm localization and its function.Methods The fragments of bcr/abl,bcr/abl-?FABD and bcr/abl-?CC gene were amplified by PCR and cloned into shuttle vector pAdTrack-CMV.The adenoviruses Ad-Bcr/Abl,Ad-Bcr/Abl-?FABD and Ad-Bcr/Abl-?CC were constructed by AdEasy system and amplified in human embryonic kidney HEK293 cells.The expression of Bcr/Abl,Bcr/Abl-?FABD and Bcr/Abl-?CC in 293 T cells were detected by the manifestation of GFP.The cytoplasm localization of Bcr/Abl,Bcr/Abl-?FABD and Bcr/Abl-?CC in 293 T cells were detected by indirect immunofluorescence with or without treatment of IM or LMB.The effects of FABD and CC domain deletion mutant on proliferation and apoptosis in293 T cell were detected by flow MTT and flow cytometry.Results After the infection of adenoviruses into 293 T cells for 24 hours,the expression of GFP can be detected.48 hours later,90% cells were packed.Bcr/Abl-?FABD protein was detected in the cytoplasm in form of particles,instead of in nuclear and the localization did not depend on the distribution of the F-actin.After being treated with IM,the distribution of Bcr/Abl was similar with Bcr/Abl-?FABD protein.Bcr/Abl-?CC translocated into nuclear.In the results of MTT,compared toBcr/Abl,effect of CC domain deletion and FABD deletion mutant on proliferation in 293 T cell was reduced(P<0.05)and by means of FCM,effect of FABD and Coiled-coil domain deletion mutant on apoptosis is promoted(P<0.05).Conclusion The adenoviruses Ad-Bcr/Abl,Ad-Bcr/Abl-?FABD and Ad-Bcr/Abl-?CC were constructed successfully.FABD didn't affect Bcr/Abl cytoplasm localization,but the distribution of Crab-?FABD mutation protein was changed.TK domain didn't influence Bcr/Abl cytoplasm localization.The deletion of Coiled-coild domain induced Bcr/Abl into the nuclear.FABD and Coiled-coil domain deletion inhibited the anti-apoptosis ability and proliferation of Bcr/Abl.