Investgation Of Recombinant Adenovirus-Mediated SH2-DED Fusion Protein Inhibit Proliferation And Induce Apoptosis Of Cml Cells

Chronic myeloid leukemia (CML)is characterized by the persistentexpression of the BCR-ABL fusion protein with constitutive tyrosine kinaseactivity. The autophosphorylated Bcr-Abl Y177recruits Grb2via its SH2domain, which is required for efficient induction of the myeloproliferativedisease by Bcr-Abl. Moreover, numerous signaling pathways, including theRas-MAPK and PI3K-Akt pathway, are activated upon the Grb2-Bcr-Ablinteraction in CML cells. The death effector domain (DED) is the criticalfactor for activation of Caspases8-induced apoptosis signal. We thusspeculated that transduction of an exogenous SH2-DED(SD) domain intothe CML cells may inhibit the binding of BCR-ABL Y177p andGrb2,activate caspase8induced apoptosis and serve as a novel CMLtreatment target.The Ad5F35system was recently reported novel recombinantadenovirus vectors that employed to deliver the exogenous SD-HA fusionprotein. SH2domain of Grb2and DED domain were subsequently cloned into Ad5F35vector, in which the HA tag was designed to facilitateimmunoblot detection and further investigate its potential effects on cellgrowth, proliferation and apoptosis in various cultured BCR-ABL positiveCML cells such as K562and KU812in vitro. Ad5F35-SD-HA wasanalyzed for its ability to efficiently infect into CML cells,to expressexogenous SD-HA fusion peptide, to induce potent anti-proliferation andapoptosis-inducing effects, and to further explore the kinase-inhibitingeffects on BCR-ABL downstream effectors. It is hoped that theAd5F35-SD-HA will exert its anti-CML effect through its function bybinding to the phospho-Bcr-Abl Y177site and competitively disruption theGrb2SH2-phospho-Bcr-Abl Y177complex. So that it can be exploited as anovel targeted molecular therapeutic strategy for CML. The mainexperiments as follows:1. The Ad5F35-SD-HA,Ad5F35-SmD-HA adenovirus expressing theSH2domain and DED domain were constructed, tested, packed andexpanded in293cells; We first assessed the infection efficiency ofAd5F35-GFP virus in K562CML cells by GFP expression analysis usingfluorescence microscopy and FCM; Then, RT-PCR was employed tofurther evaluate the expression levels of SD-HA and SmD-HA mRNA inCML cells, the expressions of both the exogenous SD-HA and theSmD-HA in K562cells were confirmed by Western blot and immunofluorescence. Moreover, expressions of both fusion proteins weredetected exclusively in the cytoplasm.2. To observe the potential specific biological effects of SD-HA bycolony formation, MTT assay, flowcytometric analysis for cell cycles, andapotosis effects assays including morphology observation andflowcytometric analysis for leukemia cell apoptosis.3. Further investgate the molecular mechanisms of the effects above.Western blot was employed to evaluate the effects of SD-HA on caspasecascade. Ad-SH2-HA efficiently infected into CML cells and functioned bypotently binding to the phospho-BCR-ABL Y177site. Ras, MAPK and Aktactivities were observed in the Ad5F35-SD-HA-treated cells.Results and conclusions were obtained as follows:1.SD-HA and SmD-HA encoding adenoviral vector were successfullyconstructed. Adenovirus with high titer was generated; Highly efficientinfection was confirmed by GFP expression analysis using fluorescencemicroscopy. The cytoplasmic localization of the SD-HA and SmD-HAfusion protein was validated by immunocytochemical staining and confocalmicroscopy. The expressions of both the wild-type SD-HA and the mutantexogenous SmD-HA in CML cells were confirmed by RT-PCR andWestern blot. The SD-HA fusion protein showed excellent biologicalactivities, thus laying a sound foundation for further evaluation of the biological functions of the SD-HA fusion peptide.2. Verified that recombinant adenovirus Ad5F35-SD-HA after infectionof CML cells have some inhibiting cell proliferation effects, such asspecificly reduced CML cells clone formation ability, and blocked cellcycle from G0/G1phase to S period. Through the cell morphologyobservation on CML cells after adenovirus Ad5F35-SD-HA infection, cellsappeared empty bubble, nuclear gather and fragment. FCM tests showedthat compared with control groups its early apoptotic cells weresignificantly increased (p﹤0.05).3. Further exploration into the underlying mechanisms revealed thatAd5F35-SD-HA infection functioned by binding to thephospho-BCR-ABL Y177site, competitively disrupted the Grb2SH2-phospho-BCR-ABL Y177complex formation, and reduced Ras,MAPK and AKT kinase activities in K562cells. Caspases8-inducedapoptosis signal could be activated by DED protein binding to DEDdomain of precursor caspases8.