Effect Of Recombinant Adenovirus Ad5F35-SH2-DED On Tumorigenicity Of K562Cells In Nude Mice

Chronic myeloid leukemia (CML) is an abnormal clonal proliferationdisease originating from the multipotent hematopoietic stem cells in whichthe BCR-ABL fusion protein with strong tyrosine kinase activity plays animportant role in the pathology. Studies showed that the Y177p ofBCR-ABL fusion protein combing with SH2domain of growth receptorbound protein2,（Grb2）after phosphorylation is a key factor，which leadsto the abnormal activation of RAS/MAPK and PI3K-AKT signal pathwayand malignant proliferation of cells. So we reason that we couldcompetitively inhibit the combination of Y177p and Grb2via exogenousSH2-DED (SD) fusion protein transduction and induce apoptosis byinvoking caspase8. This method can become an alternative for CMLtreatment.With gene recombination technology, we have built recombinantadenovirus Ad5F35-SH2-DED(Ad5F35-SD) and its mutant Ad5F35-SmD,which have been proved the inhibition of cell proliferation and the abilityof cell apoptosis induction on BCR-ABL positive leukemia cell line.. Forfurther study in vivo, we explored that SH2-DED fusion protein ability oninducing subcutaneous tumor in nude mice.Methods: 1. Preparing recombinant adenovirus Ad5F35-SD and its mutantAd5F35-SmD and carrying on amplification and titer determination ofrecombinant adenovirus Ad5F35-SD and its mutant Ad5F35-SmD.2. Constructing prevention model of recombinant adenovirus in K562cell nude mice. Prevention model observes growing situation oftransplanting tumor in nude mice by subcutaneous injection of adenovirusAd5F35-SD or Ad5F35-SmD pretreated K562cells.3. Construction of treatment model of recombinant adenovirus oftransplanting tumor in K562cell nude mice. Tumor treatment modelemploys subcutaneous injection of K562cells to establish subcutaneoustransplantation, carrying on Ad5F35-SD or Ad5F35-SmD tumor injection,to observe the growth and morphological changes of nude micetransplantation tumor, applying TUNEL method and electron microscope toobserve growth of transplanted cells.as well as with Immunohistochemistryto detecting expression of caspase-3and caspase8.The following results and conclusions are given though the aboveexperiments:1. The PCR test results showed that the recombinant adenovirusAd5F35-SD was successfully amplified, up to1.4×1012pfu/ml.2. The tumor grows slower or stops in the nude mice with Ad5F35-SDpretreatment subcutaneous transplantation of the prevention of the model,compared with the control group.3. The Ad5F35-SD treatment group significantly reduced the volumeof transplanted tumor3in the treatment model. It was observed by TUNELAd5F35-SD treatment group of subcutaneous tumor observed in nucleus isbrown and yellow; nuclear and cytoplasm concentrating, cytoplasm is redwith irregular cell shape and obvious cell apoptosis. Electron microscopyfound that Ad5F35-SD treatment group in subcutaneous tumor nuclear were irregular, chromatin marinating, with apoptotic bodies. The results ofimmunities to chemistry detection showed that the expression of apoptosisrelated proteins caspase-3and caspase-8were running up. Tumor growth issuppressed to a certain extent.4. It confirms that the recombinant adenovirus Ad5F35-SD couldinhibit K562cells in nude mice tumorigenicity and growth of transplantedtumor in nude mice, and promote tumor cell apoptosis.