In Vivo Effect Of Recombinant Adenovirus Ad5F35-SH2-Casp8in The Mouse Model Of Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is an abnormal clone proliferativedisease derived from pluripotent hematopoietic stem cells. The cause of thisdisease is the strong tyrosine kinase activity of BCR/ABL fusion protein,which can activate multiple signal transduction pathways, leading tomalignant transformation of blood cells. The177th site of tyrosinephosphorylation of BCR/ABL fusion protein can specifically bind to theSH2domain of Grb2, and then further activate RAS-MAPK, PI3K-AKT andother cell signal transduction pathways, leading to cell malignanttransformation. In the cell apoptosis pathway, the activation of apoptosisprotein Caspase8is the most critical step.Therefore, since SH2domain of Grb2can bind to the Bcr-Abl protein atthe Y177-p site specifically and Caspase8protein can induce apoptosis, weconstructed recombinant adenovirus Ad5F35-SC containing the fusion geneSH2-Casp8, which was proved the ability of promoting apoptosis in vitroand inhibiting proliferation of leukemic cells in our previous study. Here wefurther explored its in vivo effect on CML mouse model. The main methods,results and conclusions are as follows: 1. Construction and identification of the recombinant adenovirusAd5F35-SC and its mutant.SH2gene and SH2mt gene were obtained by RT-PCR and overlappingPCR, then cloned separately into the shuttle plasmid pAdT-CMV-EGFP. Theshuttle vectors were digested with PmeI and transformed intoultra-competent cells of pAd5F35-BJ5183to generate defective replicationrecombinant adenovirus vectors by homologous recombination in bacteria.After linearized by PacI digestion，these vectors were transfected intoAD293cells to package recombinant adenovirus Ad5F35-SC. PCR andwestern blot were used to identify the recombinant adenovirus, then theviruses were amplified by infecting AD293cells repeatly and virus titerswere measured.The results of PCR, restriction enzyme digestion and sequencingproved that the vectors were constructed correctly. PCR and western blotconfirmed that recombinant adenovirus Ad5F35-SC were packaged andamplified successfully, the titers of which were1.6×1012.2. The theurapic effect of Ad5F35-SC on leukemia K562cell solidtumor in nude mice model.K562cells were injected into the flanks of Balb/c nude mice toconstruct leukemia subcutaneous solid tumor model. The recombinantadenovirus Ad5F35-SC was injected subcutaneously into the solid tumorand tumor size was observed. The apoptosis of tumor cell were detected by HE histological staining, transmission electron microscopy and TUNELassay. Compared with untreated K562cells tumor group，tumor growth rateslowed down, tumor size was smaller after recombinant adenovirusAd5F35-SC treatment; Histopathology showed nuclear condensation anddeeper cell staining; TUNEL in situ apoptosis detection is also observed.Transmission electron microscopy detected apoptotic bodies and a slightswelling of mitochondria in tumor cells.These results showed that Ad5F35-SC can inhibit the growth of theK562cell subcutaneous solid tumors, induce apoptosis, and improve thesurvival state of nude mice.3. The effect of Ad5F35-SC on chronic myeloid leukemiatumorigenesis in Balb/c mice.In order to construct leukemia hematologic mouse model andprevention leukemia genesis, we injected BCR/ABL transformedBaF3-P210mice cells and BaF3-P210mice cells treated with recombinantadenovirus Ad5F35-SC to Balb/c mice through the tail vein.The Balb/c mice without any treatment were as control group. Thegrowth of mice, body weight, peripheral white blood cell count, cellmorphology of peripheral blood and bone marrow with Wright stain wereobserved; We seperated the organs of the mice of each group and observedwhether they are swelling, bleeding or getting other changes; Tissue HEstaining was performed for detecting potential pathological changes and leukemic cells infiltrate. Finally, survival curve of mice was made todetermine the survival rate changes.BP210cells transplanted mice exhibited dragged posterior limbs, lossof body weight, elevated white blood cell count; increased leukemia cells inperipheral blood and bone marrow in the4th week, as well as spleensignificantly swelled. Histopathology examination revealed that the disorderof hepatic cord and splenic nodules accompanied with infiltration ofleukemic cells. In contrast, Ad5F35-SC treated BP210cells transplantedmice had a slight decline in body weight and increased leukemia cells inperipheral blood, bone marrow smear; Liver and spleen did not swell;Histopathology HE stained showed there were minor disorder in liver cord,spleen nodules and a slight infiltration of leukemic cells. Compared with thetumor group mice, the prevention group had a significantly prolongedsurvival time in survival curve.All of the results showed that treating BP210cells with Ad5F35-SCcan significantly decrease BP210cell`s potential to cause leukemia and thenumber of leukemia cell in peripheral blood and bone marrow, reduceleukemia cell invasion of organ, and extend the lifetime of CML mice.