Transportion Of BCR-ABL Into Nucleus By Rats Induces Apopotosis And Inhibits Proliferation In K562Cells

Bcr-Abl fusion protein is the product of a reciprocal translocation t (9:22)(q34; qll) between the long arms of chromosomes9and22, which is the critical factor for the pathogenesis of chronic myeloid leukemia (CML). Bcr-Abl is only localized in cytoplasm, which significantly inhibits apoptosis and thus induces the malignant transformation of cells by activating anti-apoptotic signal. Therefore we assumed to transport Bcr-Abl into nucleus with Rapamycin analog transport system (RATS) for inducing apoptosis by the constitutive tyrosine kinase activity of Bcr-Abl.In this study, adenovirus Ad5-FLAG-3NLS-FRB*(Ad5-FN3R), wild type Ad5-HA-2FKBP-ABD (Ad5-HF2A) and mutated Ad5-HA-2FKBP-ABD (Ad5-HF2ATM) was constructed and co-infected K562cells with Rapamycin analog AP21967. The localization of the target proteins (FN3R, HF2A, HF2ATM) and Bcr-Abl was detected, as well as the consequent pro-apoptotic and anti-proliferative effects. The methods in this study are as follows: 1. The construction and identification of Ad5-FN3R, Ad5-HF2A, Ad5-HF2ATM and Ad5-null adenovirus:FLAG-3NLS-FRB*(FN3R) fusion gene was prepared from three nuclear localization signals labeled by FLAG (FLAG-3NLS) and mutation modified FRB domain of mammalian target of Rapamycin (FRB*) by overlap extension PCR. FN3R was directionally cloned into pAdTrack-CMV vector with T4DNA ligase. HA,2FKBP and ABD or ABDTM (Three amino acids important for ABD to combine Abl were mutated) were cloned into pAdTrack-CMV vector one by one. The shuttle vectors were identified by restricted digestion and DNA sequencing, then recombined with pAd5adenovirus backbone plasmid. The recombinant adenovirus was packaged and amplified in AD-293cells. The target genes in adenovirus solution were confirmed by PCR. The expression of target proteins FN3R, HF2A and HF2ATM in AD-293cells and K562cells was detected by Western blot.2. The localization of target proteins (FN3R, HF2A and HF2ATM) and Bcr-Abl in K562cells before and after RATS transporting Bcr-Abl into nucleus:K562cells were coinfected with Ad5-FN3R and Ad5-HF2A or Ad5-HF2ATM adenovirus with AP21967and the localization of target proteins (FN3R, HF2A and HF2ATM) and Bcr-Abl was detected by immunofluorescence.3. The detection of pro-apoptotic and anti-proliferative effects of Bcr-Abl into nucleus by RATS on K562cells:K562cells were coinfected with Ad5-FN3R and Ad5-HF2A or Ad5-HF2ATM adenovirus with AP21967. Cell growth was detected by MTS and the apoptosis related indicators were detected by Wright’s staining, DAPI staining and caspase3activity assay.In conclusion, we demonstrated that Ad5-FN3R, Ad5-HF2A, Ad5-HF2ATM and Ad5-null adenovirus were successfully constructed by expressing target proteins in AD-293and K562cells. Bcr-Abl was successfully transported into nucleus in K562cells coinfected with Ad5-FN3R and Ad5-HF2A adenovirus with AP21967, which was exemplified by Bcr-Abl colocating with FN3R and HF2A in the nucleus. As expected, though FN3R and HF2ATM were mainly located in the nucleus when AP21967was involved, Bcr-Abl was also in the cytoplasm of K562cells coinfected with Ad5-FN3R and Ad5-HF2ATM adenovirus. Wright’s staining, DAPI staining, caspase3activity assay and MTS assay confirmed that Bcr-Abl in the nucleus significantly induced apoptosis and inhibited growth of K562cells.